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Ch 3 Membrane Physiology

Double bond in FA tail of PL creates bend that prevents tight packing to keep phospholipids free to move around (fluidity to membrane)

Fluid Mosaic Model: Lipid bilayer with proteins embedded in it. Integral proteins extend through the membrane or are partially submerged in it. Peripheral proteins are loosely attached to the surface of the membrane. Short carbohydrate chains attach to proteins and lipids on the outer surface only.

Plasma Membrane (one of many cell membranes: ER, mitochondrial membranes, nuclear envelope, etc.) 1. Fluid Mosaic Model – lipid bilayer with proteins embedded in it (mosaic is often a piece of

art made from different materials like rock and glass) 2. Lipids – mostly phospholipids and cholesterol a. phospholipids (PL) arranged in a bilayer; amphipathic with polar and non-polar ends 1. polar head with negatively charged phosphate group (hydrophilic)

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2. two nonpolar (electrically neutral) fatty acid tails (hydrophobic) on inside of membr 3. polar head contacts water on outside and inside of cell b. cholesterol 1. binds to FA tails of phospholipids 2. prevents the fatty acid chains from packing tightly together which would decrease

the fluidity of the membrane 3. increase the structural integrity (strength) of the membrane so it doesn’t tear open 3. Membrane Proteins: Integral and peripheral a. Integral proteins (greatly outnumber peripheral proteins) 1. embedded in the lipid bilayer; part of the membrane structure and can’t be

removed without damaging the membrane 2. transmembrane proteins are integral proteins that span the entire width of the

membrane; most integral proteins are transmembrane proteins; some transmembrane proteins contain pores or channels (hollow cylinders) that water and solute can diffuse through

3. possess hydrophobic and hydrophilic regions b. Peripheral proteins are polar molecules found on the outer and inner surfaces of the

membrane and can easily be separated from it c. Lipids outnumber proteins 50:1, but proteins account for the half the mass (50%) of

the plasma membrane because they are heavier d. fluidity of the membrane allows the proteins to move around within the lipid bilayer 5. Membrane carbohydrate a. usually short-chain carbohydrates (oligosaccharides made up of 3-10 monosacs) bind to

primarily proteins and to a lesser extent lipids on the outer surface of the membrane b. the glycoproteins and glycolipids make up the glycocalyx (“sugar coating”) 6. Cytoskeleton a. Microfilaments (solid rods made of actin) – concentrated in periphery under plasma

membrane b. microtubules (hollow tubes made of tubulin) grow out from centriole located in the

centrosome; found throughout the cell c. intermediate filaments (made of a protein similar to keratin) – strongest and most

durable cytoskeletal component Functions of Membrane Proteins 1. Channels (ions and water) a. some transmembrane proteins are aquaporins (AQP) that allow water osmosis (hollow

and cylindrical) b. small, water-soluble ions (Na+, Cl-, K+, Ca2+) that can pass through ion channels 2. Carriers (transporters) a. carrier proteins are sometimes called transporters b. they bind to and transport chemicals across the membrane (glucose, amino acids, Na+,

K+) c. involved with facilitated diffusion and active transport

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3. Receptors a. protein receptors bind noncovalently to chemicals like peptide hormones and

neurotransmitters (Ach, NEpi) 1. peptides – roughly <= 50 amino acids, but often proteins with up to 200 aa’s in

length are called peptides (e.g., GH is often called either a peptide or a protein hormone with 191 aa’s)

2. proteins – usually more than 50 aa’s in length (Avg protein contains around 450 aa) b. binding triggers chemical events to occur in the cell 4. Enzymes a. membrane-bound enzymes on the outer or inner surface of the membrane that

catalyze reactions b. enzymes vary among cell types 5. Self-identity proteins (Recognition proteins) a. Proteins on the outer surface of plasma membrane along with their carbohydrate

groups are important in allowing cells to recognize “self” (normal body cells) b. MHC or “self” proteins (Major Histocompatibility Complex) 1. Made by all cells from a series of genes on chromosome 6 2. inserted into the plasma membrane c. WBC’s do not normally attack cells that they determine to be self. d. WBC’s attack non-self (bacteria, parasitic worms, viruses, fungi, bee venom, pollen

grains in the lungs) e. cells can recognize other similar cells and organize into tissues and organs, especially

during embryonic development 6. Intercellular Junction Proteins – form tight junctions, desmosomes and gap junctions

Interstitial Fluid and Cytosol 1. Interstitial Fluid (IF) - extracellular fluid (ECF) found between cells and outside of blood

vessels 2. Cytosol – fluid portion of cytoplasm (cytosol and organelles) found inside cells (intracellular

fluid). Cytoplasm is the material inside a cell excluding the nucleus. 3. ECF: IF, plasma and cerebrospinal fluid Movement of Chemicals across the Membrane: Passive and Active 1. Passive Transport Mechanisms (df gradients): df, osmosis, and fac df a. do not require ATP hydrolysis (ATP-independent) b. Not-carrier mediated 1. diffusion (df), osmosis, aquaporin channels 2. water osmoses through aquaporin channels in plasma membrane and through PL

bilayer to some extent 3. ions df through ion channels 4. O2 and CO2 diffuse through plasma membrane c. carrier-mediated transport: facilitated diffusion 2. ATP-dependent or active transport mechanisms a. require ATP hydrolysis (ATP dependent) by ATPase

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ATP + H2O -> ADP + Pi

Phosphate ion, PO4

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Phosphate group attached to

C skeleton b. energy is required to break bonds; energy is released as new bonds form in the products c. Carrier-mediated transport: active transport d. Vesicular transport 1. endocytosis (receptor-mediated, pinocytosis, phagocytosis) 2. exocytosis

Diffusion 1. substance moves down a concentration gradient from an area of high to low concentration 2. net diffusion occurs until chemicals are evenly distributed at which time there is a steady

state or equilibrium. There is random movement at equilibrium but no net diffusion 3. no ATP hydrolysis is necessary for diffusion (ATP-independent) 4. O2, CO2, FA’s and steroid hormones diffuse through the lipid bilayer

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Osmosis 1. Osmosis is the diffusion of a solvent (e.g., water, ethanol, acetone) through a

semipermeable membrane (e.g., plasma membrane); a. the membrane allows water to pass through it, but not nonpenetrating solute b. water mainly moves through water-specific channels called aquaporins (AQP)

Atomic Mass (amu): Na-23, Cl – 35, H2O - 18 c. no ATP is required for osmosis to occur (ATP-independent) 2. water molecules are strongly polar, but they are small enough to slip between the FA’s of

phospholipids, hence they can diffuse directly through the membrane or through channels 3. the FA’s of PL’s move around in the membrane creating tiny gaps between them that

water can move through by osmosis 4. water-based solution (2 parts): solvent and solute 1. solvent –water (dissolving medium) – present in greatest amount 2. solute – chemicals dissolved in the water (e.g., salt ions, glucose, amino acids) 5 free water – water molecules that are not bonded to polar molecules (H-bonds) or ions

(electrostatic attraction since opposites attract) a. the solute concentration is inversely proportional to the free water concentration b. as solute conc goes up, the free water conc goes down

[solute] 1/α [free H2O] c. polar covalent bonds (unequal sharing of e’s) result in partial charges on the surface of

molecules that dissolve in water (e.g., glucose, aa’s). H2O bind to partial charges by H-bonds. H2O electrostatically binds to charged ions (Na+, Cl-). H2O binds to other H2O

d. water molecules for hydration spheres around charged ions

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d. water molecules “stuck” to nonpenetrating solute are not free and cannot move through a semipermeable membrane by osmosis. For example, glucose, has many (5) hydroxyl (OH) groups, which increase the solubility of the molecule

6. Hypertonic and Hypotonic - solutions are always described on the basis of their [solute] a. terms used to describe two solutions separated by a semipermeable membrane

(comparative terms like tall and short, young and old) b. hypertonic 1 solution with the higher solute concentration and lower free water concentration 2. solute is nonpenetrating (can’t freely cross the membrane); water molecules bound

to solute are nonpenetrating as well 3. osmotic pressure of hypertonic solution is higher than the hypotonic solution 4. water diffuses through a semipermeable membrane into the solution with the

higher solute concentration; hypertonic solutions “attract” or “draw” water towards them

c. hypotonic 1. solution with the lower solute concentration and higher free water concentration 2. free water (unbound water) moves from a hypotonic solution through a

semipermeable membrane into a hypertonic solution d. isotonic (iso means equal) – both solutions are isotonic at the same time when their

free water and solute concentrations are equal 7. Osmotic Pressure – can be thought of as a “pulling” pressure since water moves into the

solution with the higher osmotic pressure a. pressure required to prevent water from osmosing into a solution b. measured in mmHg c. the osmotic pressure of pure water is 0 mmHg d. the greater the concentration of nonpenetrating solute, the lower the water

concentration since some H20 binds to solute (hydration sphere), the greater the pressure that must be applied to prevent the water from moving into the hypertonic solution, the greater the osmotic pressure

e. the osmotic pressure is directly proportional to the concentration of nonpenetrating solute;

[solute] 1/α [free H2O] OP α [nonpenetrating solute]

f. polar solute binds to water molecules thus reducing the concentration of free water molecules (hydration sphere)

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Relationship between solute and water concentration in a solution

Osmosis when pure water is separated from a solution containing a nonpenetrating solute

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Osmotic effects on RBC’s in isotonic, hypotonic and hypertonic solutions 1. RBC in isotonic solution – no net water movement and cell maintains its shape; steady state

solution at equilibrium 2. RBC in hypotonic solution gains water and may undergo lysis (splitting open, bursting) 3. RBC in hypertonic solution loses water and shrinks. The cell becomes spiky and wrinkled in

appearance (crenation)

Osmolarity – measure of solute concentration defined as number of osmoles of solute per liter

of solution (Osm/L) Osmole (Osm) – number of moles of solute that contribute to the osmotic pressure of a

solution 300 mOsm – concentration of osmotically active solute inside a cell

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Aquaporin (AQP)

Aquaporins (AQP)(passive, ATP-independent) 1. integral transmembrane proteins embedded in membrane that form water channels 2. Osmotic water movement through membrane phospholipids is very slow 3. Aquaporins are special water channels embedded in the plasma membrane that greatly

increase the rate of osmosis 4. channel is lined with hydrophilic amino acids that attract water molecules that move

through the channel in single file 5. 1 to 10 billion water molecules per second can pass through an aquaporin channel single

file 6. diameter of water (0.29 nm) and Na atom (0.34 nm) – water molecules are very small.

Atomic mass of H2O is 18 amu, whereas the AM of Na is 23 amu. Facilitated Diffusion B- designed to move small organic nutrients into cell, e.g., glucose and aa’s 1. passive carrier-mediated transport where carriers are proteins a. ATP hydrolysis is not required b. substance moves down a concentration gradient 2. carrier proteins span the thickness of the membrane (transmembrane integral proteins) 3. carriers can undergo reversible changes in shape (flip-flops) 4. Mechanism a. carrier protein binds to solute on side of membrane where its concentration is high;

polar solutes like glucose are hydrophilic and have partial charges on their surface and can’t diffuse through phospholipid bilayer of membrane without the aid of a carrier protein

b. carrier protein undergoes a conformational or shape change (flip flops) c. solute released to other side of cell d. carrier protein returns to its original shape (flip flops again)

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5. Facilitated Diffusion of Glucose a. glucose can’t move across the plasma membrane into body cells (e.g., muscle,

adipose, connective tissue cells) without carrier proteins; it is too large to fit through channels and it can’t diffuse through the membrane because it has partial charges on its surface due to polar covalent bonds

b. glucose moves into most cells by facilitated diffusion; this is the case for all of the aa’s as well

c. GLUT – GLUcose Transporter is the name of the carrier protein; also carries monosacs across membrane like fructose

d. glucose binds on ECF side, then transported through carrier protein as it flip flops to be released to the inside

Facilitated Diffusion

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Active transport

Active Transport (AT) 1. uses a protein carrier (transporter) to move a substance (usually an ion) across the

membrane against its concentration gradient 2. ATP hydrolysis is used to provide energy since a substance is moved against a

concentration gradient (ATP + H2O -> ADP + Pi) 3. the carrier protein has an ATPase built into its structure on cytoplasmic side of membrane;

H2O is a reactant for a hydrolase or hydrolytic enzyme 4. Two types of active transport a. Primary Active Transport – one ATP-dependent carrier protein that moves one or two

ions across membrane. The carrier protein is an ATPase that carries out ATP hydrolysis b. Secondary active transport 1. two carrier proteins a. one carrier protein is ATP-dependent and creates an ion df gradient b. the other carrier protein uses the diffusion energy of the ion gradient to move

substances against their concentration gradient 2. one of the carrier proteins splits ATP, but the other one doesn’t 3. energy stored in an ion gradient (usually Na+) to support cotransport mechanism

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Primary Active Transport (Single and Double Pumps) 1. Single Ion AT pumps: simplest examples are single ion pumps (Ca2+, H+ or proton pump) a. Ca2+ pumps are found in the axolemma of axon terminals b. H+ or proton pumps are found in the parietal cells of gastric glands 2. Double Ion AT Pumps: Primary AT Pumps that move 2 ions at the same time a. Symport pumps (symporting) – both ions move in the same direction b. Antiport pumps (antiporting) – ions move in opposite directions 3. Na+/K+ ATPase Pump (Na+/K+ Pump) – double ion antiport pump a. the carrier protein has two binding sites (one for Na+, the other for K+) and is an

antiport AT pump with an ATPase site b. pump binds to 3 Na+ and moves them to the outside of the cell (ECF) c. binds to 2 K+ and moves them to the inside of the cell (ICF) d. Mechanism of Na+/K+ Pump 1. 3 Na+ binds to the pump on the ICF side 2. Na+ binding triggers ATPase to split ATP 3. ATP hydrolysis phosphorylates the carrier protein causing it undergo a shape

change (flip flop mechanism) and release Na+ to ECF 4. K+ on the ECF side bind to carrier protein causing it to release Pi group and undergo

shape change back to original shape (flip flop) – dephosphorylation causes the pump to return to its original conformation

5. K+ released to ICF e. there are about 1 million Na+/K+ pumps in a typical neuron membrane capable of

moving 200 million ions per second

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Na+/K+ ATPase Active Transport Pump (Antiport pump)

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Secondary Active Transport (for Glucose in small intestine) 1. two carrier proteins are necessary for secondary active transport a. AT Pump: one carrier protein is a primary active transport pump with ATPase activity

that sets up an ion diffusion gradient b. Cotransporter: the second carrier protein is a cotransporter that uses the energy in the

ion gradient created by the primary AT pump to move cotransported substances across the membrane against their concentration gradient

2. Secondary Active Transport of Glucose across simple columnar epithelial cells of the small intestine

a. mechanism uses a second carrier protein called SGLT - Sodium and GLucose CoTransporter in luminal membrane that faces the intestinal lumen (also called SGLUT)

1. this is the mechanism used to absorb glucose from the lumen of the small intestine into the simple columnar epithelial cells that line the lumen

2. luminal membrane or border (facing lumen) with SGLT carriers; lumen is the open space within a tube

3. basolateral border (facing IF and capillaries) with Na+/K+ AT pumps and GLUT for facilitated diffusion of glucose

4. leakage channels for K+ in basolateral membrane or border b. SGLT cotransporters are symporters since they move 2 substances (Na+ and glucose) in

the same direction c. mechanism relies on Na+/K+ pump in basolateral membrane at the base of the cell that

faces the blood capillaries d. Mechanism 1. Na+/K+ AT pump: Primary Active Transport Pump a. Na+ are pumped out of the cell in exchange for K+ at the basolateral border by a

Primary Active Transport pump (Na+/K+ ATPase pump) b. the primary active transport antiport pump maintains a very low concentration

of Na+ in the cell that serves as a diffusion gradient for SGLT movement of Na+ (which cotransports glucose against its concentration gradient)

2. SGLT: Secondary Active Transport (glucose and Na+) a. a symport carrier protein called SGLT in the membrane that faces the lumen of

the intestine binds to Na+ and glucose b. SGLT flip flops to release both Na+ and glucose simultaneously to the cytosol c. Na+ diffuse down their concentration gradient (fac df), whereas glucose moves

against its concentration gradient by secondary AT d. the energy to move glucose against its concentration gradient comes from the

diffusion gradient for Na+ created by the primary AT pump in the basolateral membrane

e. the uptake of glucose at the luminal border occurs without ATP hydrolysis by SGLT, but is dependent on ATP hydrolysis by the Na+/K+ AT pump in the basolateral border.

f. Flip Flop 1. Na+ and glucose bind to the carrier protein causing it to change shape

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2. the carrier protein flip flops and releases both Na+ and glucose to the cytosol at the same time

g. Na+ get pumped out by the Na+/K+ pump at the other end of the cell h. glucose gets moved out by facilitated diffusion using GLUT at the other end of

the cell

Symport of Na+ and Glucose by Secondary Active Transport

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Vesicular Transport 1. large particles cross the membrane by vesicular transport 2. this is an active method of transport that requires ATP hydrolysis for energy 3. Endocytosis a. plasma membrane surrounds material outside the cell, pinches off and forms a vesicle

that moves into the cytosol b. 3 types: receptor-mediated, pinocytosis, phagocytosis 1. receptor-mediated – selective uptake of large molecules like proteins that bind to

receptors on the outer surface of the membrane a. this is the mechanism that brings LDL’s (low density lipoprotein) that carry

cholesterol into cells; cholesterol is used by cells to build and maintain the plasma membrane

b. plasma membranes typically have LDL receptors 2. pinocytosis – nonselective uptake of small solute dissolve in ECF a. this can be a mechanism to reduce the amount of plasma membrane b. exocytosis adds to the plasma membrane and pinocytosis subtracts from it 3. phagocytosis – selective uptake of solid particles outside of the cell (e.g.,

macrophages eat bacterium by phagocytosis) c. once inside the cell, lysosomes usually fuse with the vesicle and lysosomal hydrolytic

enzymes breakdown material in vesicle and release breakdown products into cytoplasm 4. Exocytosis a. membrane-enclosed vesicle forms in cell (usually at Golgi) b. secretory vesicle fuses with membrane and releases contents to outside of cell 5. Exocytosis adds to the plasma membrane and endocytosis subtracts from it

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Secretory proteins released by exocytosis

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Types of receptors and their mode of action

SIGNAL TRANSDUCTION PATHWAYS Mechanisms by which Chemical Messengers (hormones and neurotransmitters) act on Target

Cells 1. Signal Transduction a. Signal – hormone (e.g., amines like Epi, peptides like INS and GH, steroid hormones like

E and P and T) or neurotransmitter (e.g., ACh) that binds to a receptor b. Transduction – Signal stimulates or causes a cellular response c. Signal transduction is when a signal molecule 1. binds to a receptor on the plasma membrane or inside the cell and 2. triggers chemical reactions to occur inside the cell that results in a 3. cellular response (e.g., membrane transport, contraction of muscle cell, secretion,

change in metabolism, gene expression to make a protein) d. A signal transduction is a cascade or series of chemical reactions inside a cell triggered

by signal-receptor binding 2. Signal transduction pathways involve a SIGNAL CASCADE where 1 event triggers another

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3. Two types of Signals: Lipophilic (lipid soluble) and Lipophobic (water soluble) – baed on the solubility of the signal in the phospholipid bilayer of the plasma membrane

a. LIPOPHILIC (Hydrophobic): Lipid-soluble signals (substances) like steroid hormones that enter cells and bind to protein receptors inside the cell. DIFFUSE through plasma membr. RECEPTORS inside cell

b. LIPOPHOBIC (Hydrophilic): Water soluble signals (substances) like protein hormones or neurotransmitters that bind to protein receptors on the plasma membrane. DO NOT DIFFUSE through plasma membr. RECEPTORS on plasma membrane

Ligand 1. molecule that binds to another molecule 2. Substance that forms a complex with a biomolecule to serve a biological purpose 3. Example: Nt’s or hormones are ligands when they bind to protein receptors Steroid Hormones (Lipophilic or hydrophobic) 1. Lipid-Soluble Steroid Hormones: Lipophilic (lipid-soluble, hydrophobic) extracellular

messengers

Testosterone

a. steroid hormones made from cholesterol (polycyclic aromatic rings); they are hydrophobic or lipophilic. Steroid hormones include estradiol, progesterone, testosterone, aldosterone, cortisol, DHEA

a. also mechanism for lipid-soluble thyroid hormones (T3, T4) and nitric oxide (NO) b. thyroid hormone moves into cells by facilitated diffusion 2. steroid hormone enters the cell by dissolving into and moving through the lipid bilayer

by diffusion 3. steroid hormone binds to a protein receptor in the cytosol or nucleus that acts as a

ligand-dependent transcription factor a. Steroid Receptor = Steroid-activated transcription factor = HRC or Hormone

Receptor Complex 1. the steroid receptor is an inactive transcription factor protein inside the cell

that is considered to be an “inactive” transcription factor since it isn’t capable of binding to DNA.

2. Transcription factors are proteins that bind to specific DNA N-base sequences in the promoter region of gene, thereby controlling gene activity.

3. Activator or Repressor: They usually turn a gene on (activator) to stimulate RNA polymerase to make mRNA (they can also turn a gene off (block or repressor) to inhibit gene transcription)

Hormone binds to RECEPTOR Inactive Transcription Factor (RECEPTOR) -------------------------→ HRC (active transcription factor)

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b. Hormone-Receptor Complex (HRC) = Active transcription factor 1. the binding of a steroid hormone to the inactive transcription factor causes the

transcription factor to undergo a conformational change that activates it to become an “active” transcription factor.

2. An activated transcription factor is capable of binding to DNA 3. the active transcription factor (hormone-receptor complex or HRC) then binds

to the promoter site of a protein-encoding gene c. binding of an HRC to a gene generally turns the gene “on” (Activator) or turns the

gene “off” in some cases (Repressor) d. An activated protein-encoding gene that has been turned “on” makes mRNA, hence

a protein e. The protein (e.g., enzyme) produced by transcription-translation elicits the

hormone’s effect on the cell

Hormone Receptor: DNA- binding site binds to promoter site on gene; Steroid hormone binds to hormone binding site, thus activating transcription factor to form hormone-receptor complex (HRC). The receptor may interact with

chemicals other than the hormone that affect the HRC activity

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LIPOPHOBIC SIGNALS: Peptide Hormones and Neurotransmitters (water-soluble, Hydrophilic) 1. Neurotransmitters (e.g., Ach) that open (or close) gated ion channels 2. Tyrosine kinase pathway (e.g., insulin) 3. Second messenger pathways: a. cAMP (most common system) b. Phosphoinositol System (DAG-IP3-Ca2+) Peptides and Proteins 1. Both are made from chains of amino acids held together by peptide bonds 2. Peptides contain 2-50 aa’s, while proteins contain 50 or more aa’s. This is an arbitrary

cutoff. They include dipeptides, tripeptides and tetrapeptides, etc. Growth hormone is made up of 191 amino acids and is considered to be a peptide hormone, altho it is also referred to as a protein hormone.

3. The average protein contains around 500 aa’s. 4. Small proteins up to around 200 aa’s are often called peptide hormones (e.g., GH, INS,

FSH) Peptide Hormones (all of the hormones below would typically be called peptide hormones)

hormone aa hormone aa OXT ADH INS GH

8 9

51 191

PRL FSH LH

198 207 212

Allosteric modulation (modification) 1. Chemical binds to non-substrate site on an enzyme that affects the affinity (increase or

decrease) of the active site for substrate a. allosteric activation – increase catalytic activity of enzyme (“turn on”) b. allosteric inhibition – decrease catalytic activity of enzyme (“turn off”)

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2. Most enzymes affected by allosteric modulation 3. Most important allosteric modifier of enzyme activity is phosphorylation and

dephosphorylation. This usually a shape or structural change in the enzyme. 4. common type of modulation is the use of a kinase to activate an enzyme by phosphorylating

its allosteric site Neurotransmitters like Acetylcholine ( ACh) that open (or close) ion channels in membrane - Hydrophilic (Lipophobic) external messenger binds to membrane receptor that open or close ion channels 1. SIGNAL = External messenger (e.g., neurotransmitters like ACh) binds to a membrane

receptor 2. this opens up a chemical “gate” so that ions can move through an ion channel 3. the ions moving through the channel trigger a cellular response 4. 3 types of gated channels: chemical-gated, voltage-gated, mechanical-gated

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Tyrosine kinase pathway used by Insulin

Tyrosine Kinase Pathway (used by Insulin) 1. External messenger like Insulin (INS) – peptide hormone (51 aa) secreted by pancreatic beta

cells 2. INS binds to Tyrosine Kinase Receptor (which has enzyme activity) – receptor protein has a

number of tyrosine aa’s a. the receptor is both a hormone receptor and an enzyme b. kinase – enzyme that transfers a phosphate group from ATP (or another high energy

donor) to specific substrates in a process called phosphorylation (allosteric modulation) 3. INS binding to the receptor on the ECF side activates its enzymatic site on the ICF side of

the membrane 4. the active enzyme, tyrosine kinase, then phosphorylates proteins (e.g., enzymes) to

allosterically activate them inside the cell. Turning on the enzyme brings about the cell response (e.g., incr protein syn, incr glycogen synthesis, incr facilitated df of glucose into cells)

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General reaction catalyzed by kinase

cAMP Second Messenger pathway: Hydrophilic SIGNAL (External Messenger) 1. cAMP (cyclic adenosine monophosphate) is the most common second messenger 2. SIGNAL: first messenger (e.g., peptide hormone or neurotransmitter) binds to GPCR

membrane receptor on ECF-side a. examples of first messengers: FSH, LH, TSH, ACTH, CT, hCG, glucagon (GCG), Ach

(Muscarinic 2, M2, receptors), Epi (α2, β1, β2,) receptors b. GPCR – G-Protein Coupled Receptor c. GPCR is associated with a membrane protein called G protein (guanosine nucleotide

binding protein) 1. G protein is a protein with GTPase activity that is activated by GPCR 2. once activated by GPCR, GTP binds to G proteins and is then hydrolyzed to GDP + Pi.

This activates the G protein. 3. The GTP-activated G protein then activates adenylyl cyclase (AC) to generate cAMP 3. binding of peptide hormone to GPCR activates the associated G protein (membrane

protein on ICF side) on the inside of the membrane. G protein is in the membrane and consists of alpha (α), beta (β) and gamma (γ) subunits

a. the G protein is the transducer (signal transduction pathway) since it converts the info in the first messenger to the same info in the second messenger inside the cell

b. the G protein is made up of 3 subunits: alpha (α), beta (β), and gamma (γ) c. G proteins are so named because they are proteins bound to a guanine-containing

nucleotide (either GTP when active or GDP when inactive) 4. Allosteric modulation: the alpha (α) unit of the G protein then activates the enzyme

adenylyl cyclase on the cytoplasmic side of the membrane. Adenylyl cyclase (AC) is the effector protein in the membrane on the ICF side

a. adenylyl cyclase converts ATP to cAMP by cleaving off two phosphates 5. cAMP is the intracellular second messenger (the peptide hormone is the extracellular first

messenger) a. one could inject a solution of cAMP into the cell and observe the hormone’s effect 6. cAMP triggers a series of biochemical events that ultimately bring about the cell’s response

to the hormone a. cAMP binds to and activates protein kinase A (PKA) b. protein kinase A phosphorylates preexisting intracellular proteins (often enzymes) c. phosphorylation activates the enzymes by changing their shape (allosteric modulation) d. the activated enzyme triggers the cellular response by triggering reactions in the cell 7. cAMP is degraded in cells by phosphodiesterase, a cytosolic enzyme that is continuously

active

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8. Activating the cAMP pathway can a. modify heart rate (Ach, NEpi) b. stimulate the synthesis of estrogen by ovarian cells (FSH) c. stimulate hepatic glycogenolysis (glucagon) cAMP 2nd Messenger System (cyclic adenosine monophosphate). The first messenger are

chemicals like Epi and peptide hormones like FSH, LH and glucagon

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Phosphoinositol System (DAG and IP3 are Second Messengers in this System) 1. some cells use IP3 and DAG as second messengers instead of cAMP 2. First messenger (hormone or other chemical like Ach) binds to GPCR (G-protein coupled

receptor, transmembrane protein) a. examples: Oxytocin (OT), GnRH, Epi (α1 receptors) b. ACh (M1 and M3 receptors) 3. this stimulates the α unit of the G protein associated with the receptor to activate an

enzyme called Phospholipase C (effector protein) that is found in the plasma membrane on the cytosolic side. G proteins consist of alpha, beta, and gamma subunits.

4. the enzyme (phospholipase C) breaks down PIP2 (phosphatidylinositol biphosphate) which is a component of the fatty acids of the phospholipids in the membrane

a. PIP2 -> DAG + IP3 (DAG and IP3 are the second messengers) b. DAG – diacylglycerol (DAG is lipid-soluble and remains in the membrane) c. IP3 – inositol triphosphate 5. IP3 is water soluble and diffuses into the cytosol 6. IP3 binds to IP3 receptors on gates that control Ca2+ channels in the tubular cisterns of

smooth ER membrane 7. this leads to an increase in the [Ca2+] of the cytosol 8. Ca2+ bind to and activate calmodulin (CaM), an intracellular Ca2+ binding protein 9. Activation of calmodulin is similar to activation of protein kinase A by cAMP 10. Calmodulin bound to Ca2+ (CaM-Ca2+) usually activates intracellular enzymes that

phosphorylates proteins (e.g., kinase) a. cAMP pathways activate protein kinase A b. IP3 pathway activates calmodulin to activate a kinase (Ca2+/CaM-dependent kinase) c. kinase – enzyme that transfers a phosphate group from ATP (or another high energy

donor) to specific substrates in a process called phosphorylation 11. the enzymes activated by CaM-Ca2+ complex then trigger the cell response to the first

messenger a. Effects of increasing Ca2+ concentrations that lead to calmodulin activation 1. muscle contraction 2. exocytosis of neurotransmitters from axon terminus 12. DAG Pathway (occurs at same time as IP3 effects) a. DAG sets off another second messenger pathway where DAG is the second messenger b. DAG activates protein kinase C (PKC) by binding to it c. PKC (bound to DAG) phosphorylates inactive proteins in the cytosol, thus activating

them d. the now active proteins produce another cellular response

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Phosphoinositol System (DAG-IP3-Ca2+ Second Messenger System)

Mechanism of action of hydrophilic hormones via activation of IP3-Ca2+ Second Messenger

System

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Examples of Second Messenger Systems

cAMP System

Phosphoinositol

system cGMP System

Tyrosine kinase

system

Ligand:

Neurotransmitters

(Receptor)

Epinephrine (α2, β1, β2)

Acetylcholine (M2)

Epinephrine (α1)

Acetylcholine (M1, M3) - -

Ligand:

Hormones

ACTH, ANP, CRH, CT,

FSH, Glucagon, hCG,

LH, MSH, PTH, TSH

AGT, GnRH, GHRH,

Oxytocin, TRH ANP, Nitric oxide

INS, IGF,

PDGF

Transducer Gs (β1, β2), Gi (α2, M2) Gq - -

Primary effector Adenylyl cyclase Phospholipase C guanylate cyclase receptor tyrosine kinase

Secondary messenger cAMP (cyclic adenosine

monophosphate)

IP3 (inositol 1,4,5

trisphosphate) and DAG

(Diacylglycerol), both

from PIP2

cGMP protein phosphatase

Secondary effector protein kinase A

Ca++ release (see calcium-

binding protein) and PKC

(protein kinase C)

protein kinase G -