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BMB 462: EXAM 1

What are lipids?
• The cellular components that are soluble in organic solvents (chloroform, methanol) but sparingly soluble or insoluble in water
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General Properties of lipids
• One of the 4 major groups of biomolecules but unlike proteins, nucleic acids and polysaccharides they are NOT polymers • Chemically the most diverse group • A catch all category of things that are hydrophobic and sparingly soluble in water (but soluble in organic solvents)
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Functions of lipids
• Form bilayers which along with proteins are major components of membranes • Energy stores-triacylglycerols (fats and oils) • Intercellular and intracellular signaling molecules • Many other functions as well
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Structure of fatty acids
• Carboxylic acids with long hydrocarbon chains • Saturated • Unsaturated
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Properties of Fatty acids
• Amphipathic • Form micelles in aqueous solution • Most have even carbon # • Double bonds are almost always cis Cis kinks the molecule Trans double bonds are rare in nature and don't kink the molecule • Multiple double bonds are almost always three carbons apart, i.e. not conjugated
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Functions of fatty acids
• One of the building blocks of some lipids • Signaling
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Structure of triacylglycerols
• A glycerol backbone with three fatty acids attached by ester linkages • The fatty acids can be the same but are most often different.
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Properties of triacylglycerols
• Most abundant lipids in the human body but DO NOT form membranes. • Hydrophobic - stored in anhydrous form • F.A.s attached determine properties (liquid or solid at RT)
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Functions of triacylglycerols
• Primarily metabolic storage molecules (fats in animals, oils in plants). Provide ~6 times the metabolic energy of carbohydrates Glycogen stores last 24 hrs -TAG stores last 2-3 months! • Insulation • Normal man 21% fat, normal woman 26% fat.
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General Properties of of Membrane Lipids
• Membrane lipids are amphipathic • Form bilayers and liposomes in aqueous solutions
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Key Concept of Membrane Lipids
- polar head group & some form of backbone - hydrophobic tails - properties of lipids are controlled by the parts (head & tail)
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Classes of membrane lipids
glycerophospholipids -galactolipids & sulfolipids -sphingolipids -cholesterol -phospholipases
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Glycerophospholipids Structure
» glycerol backbone with FA’s esterified to C1 and C2 and a head group attached to C3 by a phosphodiester linkage. » some have ether linkage to C1 » enormous diversity of structure
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Properties of Glycerophospholipids
» different fatty acids alter properties » C1= often saturated » C2= often mono- or polyunsaturated » different head-groups alter properties
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Function of Glycerophospholipids
» major membrane constituent » precursor for signal molecules
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Structure of Galactolipids & Sulfolipids
» Glycerol backbone with FA’s esterified to C1 and C2 and a head group attached to C3 by a glycosidic linkage.
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Properties of Galactolipids & Sulfolipids
» Similar to glycerophospholipids
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Function of Galactolipids & Sulfolipids
» Abundant in plant chloroplasts » Most abundant membrane lipids on the planet » May compensate for phosphate limitation in plants
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• Structure of Sphingolipids
Built on the 18 carbon amino alcohol sphingosine. » amino group on C2 » hydroxyl group on C3 » trans double bond A fatty acid is attached by an amide bond to the nitrogen on carbon 2 of sphingosine backbone with a polar head group attached to C1.
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• Properties of Sphingolipids
Similar to glycerophospholipids and galactolipids
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• Function of Sphingolipids
Prevalent in myelin and neuronal membranes Cell recognition (ABO blood groups)
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• Structure of Cholesterol
Rigid, 4 ring, planar structure Hydrocarbon Tail OH on C3
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Properties of Cholesterol Amphipathic
» Hydrocarbon rings and tail are hydrophobic » OH is polar Rigid w/ cholesterol than without
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• Functions of Cholesterol
decreases Membrane Fluidity Signal Precursor Bile Acid Precursor which helps digest lipids
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Cholesterol is an?
isoprenoid
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• Phospholipases are?
– enzymes that hydrolyze bonds in lipids
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Specificity of Phospholipases
• Phospholipase A1 (PLA1) • Phospholipase A2 (PLA2) • Phospholipase C (PLC) • Phospholipase D (PLD)
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• Phospholipase A1 (PLA1) cleaves
between FA and carbon 1 of glycerol
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Phospholipase A2 (PLA2) cleaves
between FA and carbon 2 of glycerol
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• Phospholipase C (PLC) cleaves
between carbon 3 of glycerol and phosphate
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• Phospholipase D (PLD) cleaves
between phosphate and head group.
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Functions of Phospholipases
• Digestion • Lipid Turn Over • Signaling
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Key Concept of Phospholipases
Metabolism of lipids produces signals
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What are the Hydrolysis products of membrane lipids
PLC PLA2
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What is PLC
PIP2 is in inner leaf of plasma membrane. PLC activated by hormones Releases IP3 and DAG, both of which are signals!!
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What is PLA2
Releases arachidonate which is processed to eicosanoids » Local (paracrine) hormones » Site of action localized and very brief half life (seconds to minutes)
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A Leukotriene does what?
Causes smooth muscle contraction (Asthma)
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• what does Cox function have to do with PLA2
COX function is required for prostaglandin and thromboxane formation
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» » COX is the site of?
action for non-steroidal antiinflammatory drugs (NSAIDs): aspirin, Tylenol, ibuprofen, Celebrex, Vioxx.
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COX-1 and COX-2 are?
isozymes (~65%identical).
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COX-1 produces?
prostaglandins involved in controlling gastric mucin secretion.
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COX-2 produces?
prostaglandins involved in pain, inflammation and fever.
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Celebrex, Vioxx etc. were designed based on?
differences in COX-1 and COX-2 crystal structures. Bind ~1,000X better to COX-2, so greatly reduced stomach problems. It also turns out they increase the risk cardiovascular problems.
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• COX inhibition types
1. Irreversible 2. Reversible » Nonspecific » Specific
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Steroids are
endocrine hormones • Potent signaling molecules derived from cholesterol • Circulate throughout the body • Control diverse bodily functions
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Isopentenyl pyrophosphate (IPP), which is a activated isoprene, is a
key intermediate in the synthesis of a wide variety of compounds.
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Structure of Other lipids derived from isoprene
-repeating isoprene sequencing
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Properties of Other lipids derived from isoprene
- all hydrophobic
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Function of Other lipids derived from isoprene
Various function from various compounds
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Main point of Other lipids derived from isoprene
Isoprene is used to make tons of compounds w/ many different function
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Two ways that we can Work With Lipids?
Isolation using organic solvents. Separation using absorption chromatography or TLC.
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Explain Separation using absorption chromatography or TLC.
-stationary phase - mobile phase – starts out non-polar and increases in polarity
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When working with lipids, how do you Determine the chain length and saturation by?
GLC or HPLC.
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When working with lipids, how do you Determine the fatty acid position by?
enzymatic degradation.
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When working with lipids, how do you Determine the double bond positions by?
mass spec.
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Carbon skeleton:12:0
Structure: ch3(ch2)10COOH Systematic name: n –dodecanoic acid Common name: lauric acid MP: 44.2 C
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Carbon skeleton: 18:0
Structure: ch3(ch2)16COOH Systematic name: n-octadecanoic acid Common name: Steric acid MP: 69.6 C
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Carbon skeleton:14:0
Structure: ch3(ch2)12COOH Systematic name: n-tetradecanoic acid Common name: myristic acid MP:53.9 C
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Carbon skeleton: 20:0
Structure: ch3(ch2)18COOH Systematic name: n-eicosanoic acid Common name: Arachidic acid MP: 76.5 C
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Carbon skeleton: 16:0
Structure: ch3(ch2)14COOH Systematic name: n- hexadecanoic acid Common name: palmitic acid MP: 63.1
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Carbon skeleton: 24:0
Structure: ch3(ch2)22COOH Systematic name: n-tetracosanoic acid Common name: Lignoceric acid MP: 86.0 C
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Carbon skeleton: 16:1 (9)
Systematic name: cis-9-hexdecenoic acid Common name: Palmitoleic Acid MP: 0.5 – 1 C
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Carbon skeleton: 18:1 (9)
Systematic name: Cis-9-Octadecenoic acid Common name: Oleic Acid MP: 13.4
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Carbon skeleton:18:2 (9,12)
Systematic name: c-c-9,12-octadecadienoic Common name: linoleic Acid MP: 1 – 5 C
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Carbon skeleton: 18:3 (9,12,15)
Systematic name: c-c-c-9,12,15-octadecatrienoic acid Common name: Alpha Linolenic acid MP: -11
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Carbon skeleton: 20:4
Systematic name: c-c-c-c-5,8,11,14-Icosatetraenoic acid Common name: Arachidonoic acid MP: -49.5 C
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The longer the chain length in FA, ? melting point
higher the melting point
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the more double bonds in a FA, the ? the Melting point
lower the melting point
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Functions of biological membranes.
– Selective permeability barrier with channels – Demarcate cell boundaries and compartments. – Contain specific receptors for external stimuli – Generate signals --- electrical and chemical
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• Common features despite diversity of biological membranes.
– Membranes are sheet-like structures 6- 10 nm (60-100 Å) thick. – Membrane lipids are amphipathic, i.e., they posses both hydrophobic and hydrophilic components, arranged as lipid bilayers. – Membranes are not covalently assembled but are linked by many noncovalent interactions. – Membranes are fluid structures. – Membranes can fuse and self seal. – Membranes consist of polar lipids and proteins to which carbohydrates are often attached. – Proteins serve as pumps, channels, receptors, energy transducers and enzymes. – Membranes are selectively permeable. – Membranes are asymmetric – Membranes are usually polarized electrically.
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• Lipid Composition of plasma membranes
– Different Organisms & Cell Types – Different Organelles
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• Lipid Composition of bilayer plasma membrane
– Inner and Outer Leaflets – Asymmetry
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• Fluid Mosaic Model is?
2D array of lipids and proteins (2 major components)
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Components of Fluid Mosaic Model
-lipids (which is needed to hold proteins), proteins, and carbs (linked to lipids and proteins)
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– Movement of Fluid Mosaic Model
a lot of lateral movement, transverse movement is limited
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What (X) is mainly inside the lipid bilayer?
-ethanolamine, -serine, -linositol, -linositol-4-phosphate, - linositol-4,5-biphosphate, phosphatidic acid
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What (X) is mainly outside the lipid bilayer?
-choline, sphingomyelin
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– 3 Types of Membrane Proteins
• Peripheral • Integral and Lipid anchored (FA/isoprenoid & GPI linked)
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What are Peripheral membrane proteins?
-Held in place by electrostatic or hydrogen bonds -Polar like interactions -removal results in a functional protein - can be removed by changing pH and/or salt
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2 Types of integral Membrane Proteins
– α-helical – β-barrel
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Define integral Membrane Proteins
- held in place by hydrophobic interactions - not functional if removed -can be removed by adding detergents or organic solvents – Asymmetry
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What are α-helical integral membrane proteins?
One way integral MP can get into a membrane if it does use hydrophobic residues (Val, Luc,Isoleu.)
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What are β-barrel integral membrane proteins?
» 20 or more transmembrane segments form beta-sheets that line a cylinder. » Alternating amino acid are hydrophobic/hydrophilic: face bilayer/ line the cylinder.
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Define Lipid Anchored membrane proteins?
- A covalent bond btwn a lipid & an A.A in a protein – Asymmetry
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What are 3 types of lipid anchored membrane proteins?
» Fatty acid linkages » Isoprenoid linkages » GPI linked (glycosylated derivatives of phosphatidylinositol)
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FA linkages of lipid anchored MP have what type of bonds?
-theoester or ester bond btwn F.A (palmitate; 16:0) & Ser or Cys
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How do you remove a lipid anchored MP?
Either break covalent bond or destroy membrane
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What happens if the temp. is altered in Gel vs. Fluid State of MP?
If you increase temp., gel will become liquefied.
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What is the lipid composition of the Gel vs. Fluid State of MP
In the gel state nothing moves, as temp increases, the gel becomes more fluid and achieves order, lateral movement but not much roation in acy tails With increased heat, liquid becomes disordered, there is a lot of rotation, and lateral/transverse movement
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Transverse Diffusion in the Dynamics of Membrane Lipids
–Energetics – must move polar head group thur hydrophobic core. Flippases are enzymes that catalyze movement and allows the polar head group move thur the hydrophobic core quickly.
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Lateral Diffusion in the Dynamics of Membrane Lipids
– Energetics – Hop diffusion – molecular fences control lipids which tend to jump the fence into another protein corral – Protein Corrals
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• What is involved in Lateral Diffusion in Membrane Lipids?
Sphingolipid/Cholesterol Micordomains (Rafts) - Caveolin
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What are Sphingolipid/Cholesterol Micordomains (Rafts)
» Composition – long Sat. tails - cholesterol is rigid and fairly long (GPI linked outside, FA linked inside)
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» What is the Sphingolipid/Cholesterol Micordomains (Rafts)Function?
– Clusters proteins w/ similar function
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What are Caveolin?
Aka little caves, found in some special microdomains » Structure – an integral membrane protein with two globular domains connected by a hairpin shaped hydrophobic domain, which binds the protein to the cytoplamic leaflet of the PM. Three palmitoyl groups are attached to the carboxyl-terminal globular domain
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What is Caveolin function's?
» Function – binds cholesterol in the membrane, and the presence of caveolin forces the associated lipid bilaer to curve inward, forming little caves
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What 8 Biological Functions require membrane function?
Budding of vesicles from golgi complex, exocytosis, endocytosis, fusion of endosome and lysosome, viral infection,Fusion of sperm and egg, fusion of small vacuoles (plants), and separation of two plasma membranes at cell division
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What happens when when membrane fusion is mediated by membrane proteins?
Bring together membranes, help disrupt bilayer, regulates
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Membranes as Selectively permeable Barriers Example: Cystic Fibrosis energetics & Activation
• Energetics Very small non-polar molecules can cross bilayer w/o transporter – Activation Barrier lowers to transport molecules – Electrochemical Potential is which direction the solute moves spontaneously
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– Passive vs. Active - Selectively permeable Barriers
Passive is going with he electrical or chemical gradient Active requires energy input from somewhere else
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• what are 4 Types of passive Membrane Transporters (Based on Energetics)
– Simple Diffusion – Facilitated Diffusion – Ion Channels – Ionophores
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What is Simple Diffusion?
Non-polar compounds only Travels down concentration gradient
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What is Facilitated Diffusion (aka passive transport)
-requires protein still moves down electrochemical gradient
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What are Ion Channels
-subcatagory of facilitated diffusion -can be gated (regulated)
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What are Ionophores
-hydrophillic inside -hydrophobic outside -passive -can kill a cell
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Transporters can be based on?
Energetics Properties Solute # and direction of movement
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What are the active transporters?
–Primary – always uses ATP as energy – Secondary – uses energy from 1 solute moving down (exogonic) its gradient to move a 2nd solute against (endogonic – uses energy) its gradient
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3 types of primary (active) transporters?
- P type – ATPases -F type or V type – ATPases - ABC transporters
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What are two transports based on Transport Properties
• Channels • Carriers
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What is a channel?
Rate approaches unhindred diffusion (10^8) Never Active Transport Less sterospecfic Pore in membrane (regulated)
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What is a carrier?
-enzyme like kinetics -can be saturated (limited to stop at saturated) -rate is below diffusion -more sterospecific
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What pushes molecule across membrane in active transport?
Charge or concentration of solute
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What are the 2 catagories of transport based on Solute # and Direction of Movement?
• Uniport • Cotransport – Symport – Antiport
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what is H+ across mitochondria
inside: Low outside: High
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what is H+ across bacterial
inside: Low outside: High
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Na+ across animal
inside: Low outside: High
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K across animal
Inside: High Outside: Low
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Ca+ across ER, SR, & plasma Membrane
inside: Low outside: High
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The GLUT family is what type of transporters?
Membrane transport
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What is the structure and function of The GLUT family transporters?
– Structure – 12 transmembrane helices (amphiphatic helices) – Function – To move glucose down it’s gradient
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What are the kinetics of GLUT family of transporters?
Vm ~> point of saturation Kt ~> half the rate of Vmax
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What is the location and function of GLUT 1?
RBC / transport energy to RBC
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What is the location and function of GLUT 2?
Liver & S. Intestines / Moves glucose out of the liver
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What is the location and function of GLUT 4?
Muscles & Fat tissue / Controlled by insulin
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What is the Classification of GLUT family transporters?
MSF transporters
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P-type active transport structure and function
• Structure – an integral protein w/ 10 predicted membranes spanning region in a single polypeptide, some have a second subunit; widely distrubuted • Function – takes (3) Na+ from inside the cell and takes it outside and picks up 2 K+ molecules and drops them inside the cell
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What is an example of P-type active transport?
Na+/K+ ATPase – Na+ and K+ ions moved against electrochemical gradients -High K inside cell -High NA outside of cell – This gradient provides the energy for many (but not all) secondary active transport.
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What is the structure & function F-type and V-type ATPase
• Structure - F0 – rotates as typically H+ moves across membrane - F1 - Catalysts • Function - ATP synthase - Can be used to change the pH of plant vacuoles - Can change pH @ golgi, lysosomes
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What is ABC transporters?
ATP binding Cassette - A large family of ATP-dependent transporters that pump A.A, peptides, proteins, metal ions, various lipids, bile salts, and many hydrophobic compounds, including drugs, out of cells against a concentration gradient.
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What is the structure of ABC transporters
• Structure - All ABC transporters have two nucleotide-binding domains (NBDs) and two transmembrane domains (Fig. 11–41). In some cases, all these domains are in a single long polypeptide; other ABC transporters have two subunits, each contributing an NBD and a domain with six (or in some cases ten) transmembrane helices. Although many of the ABC transporters are in the plasma membrane, some types are also found in the endoplasmic reticulum and in the membranes of mitochondria and lysosomes. Most ABC transporters act as pumps, but at least some members of the superfamily act as ion channels that are opened and closed by ATP hydrolysis.
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What is the function of ABC transporters?
-primary active carriers – ex. Drug pumping meds out of a cell -works against gradient -As a channel, nucleotide binding regulates flow
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Secondary Active Transport is either?
Antiport or symport
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What is a example of secondary active transport?
Lactose Permease (aka Lac Y)
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What is the function of lactose permease (2nd active transport)?
Bring lactose into the cells
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What is the Energy source lactose permease (2nd active transport)?
H+ across plasma mebrane
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What is the Classification of lactose permease (2nd active transport)?
-2nd active transport - symport - carrier
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What is the function of the Na+/Glucose Symporter?
Moves glucose from diet into intestinal cell
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What is the Energy Source of the Na+/Glucose Symporter?
Na+ gradient
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What is the Classification of Na+/Glucose Symporter
-2nd active transport -symporter -channel
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What are the 4 General features of signal transduction systems
-specificity, amplification, Desensitization, Integration
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What is specificity?
– Specificity - Signal molecule fits binding site on its complementary receptor;other signals do not fit.
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What is Amplification?
When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade.
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What is Desensitization -
Receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface.
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What is Integration -
When two signals have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm, the regulatory outcome results from the integrated input from both receptors.
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What is the General Process of Signal Transduction?
– Signal (1st Messenger) - either hormone, from environment, or voltage – Receptor – some type of protein – intergral or soluble protein – 2nd Messenger(s) - diffusable – Phosphorylation Cascades – enzyme 1 adds *P* to enzyme 2 & becomes active, E2 to E3 that becomes activated – Effector(s) – change of enzyme activity or gene expression – Cellular response – either a change in metabolism, gene expression, or membrane potential – Signal Termination – degrading primary signal (1st signal), degrade 2nd messenger (Ca or cAMP), dephosphorylate the cascade
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What are the Six general types of receptors of Signal Transduction
– Gated Ion Channels – Receptor Enzymes – G protein-coupled Receptors – Steroid Receptors – Receptors with no intrinsic enzyme activity – Adhesion receptors
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What are Gated Ion Channels?
Gated by a ligand or voltaged gated Opens or closes in response to concentration of signal ligand (S) or membrane potential.
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What are Receptor Enzymes
1 TM alpha helix function by dimerlzation actives an enzyme in the cytosol domain common receptor: tyrosine kinase Ligand binding to extracellular domain stimulates enzyme activity in intracellular domain.
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What are G protein-coupled Receptors
Ligand binding changes affinity of the receptor for a G-protein 7 TM alpha helix (aka serpentine receptor) External ligand binding to receptor (R) activates an intracellular GTP-binding protein (G), which regulates an enzyme (Enz) that generates an intracellular second messenger, X.
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What are Steroid Receptors
(aka hormone receptor) ligand bind to cytosolic or nucular protein that changes gene expression Steroid binding to a nuclear receptor protein allows the receptor to regulate the expression of specific genes.
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What are Receptors with no intrinsic enzyme activity
Interacts with cytosolic protein kinase, which activates a gene-regulating protein (directly or through a cascade of protein kinases), changing gene expression.
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What are Adhesion receptors
Binds molecules in extracellular matrix, changes conformation, thus altering its interaction with cytoskeleton.
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Neuronal Signaling (Gated Channels)Process
– Signal – change in membrane potential or neurotransmitter – Receptor – gated channel – 2nd Messenger – Ca 2+ & Na+ – Cellular Response – Na+ enters cell and K+ leaves cell
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• Signal Transduction - Insulin Regulation of Gene Expression (Receptor Enzymes)
– Signal Insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues. – Receptor Insulin receptor phosphorylates IRS-1 on its Tyr residues. – Phosphorylation Cascade - SH2 domain of Grb2 binds to P –Tyr of IRS-1. Sos binds to Grb2, then to Ras, causing GDP release and GTP binding to Ras. - Activated Ras binds and activates Raf-1. - Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and a Tyr residue, activating it. – Effector ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them. – Cellular Response Change in gene expression Phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell division.
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insulin also mediates other cellular effects through branches in the signaling pathway like?
Conformational Changes in the Insulin
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What are the steps in β-Adrenergic Receptor (G proteincoupled receptors) Signal Transduction?
– Signal - epinephrine – Receptor – G-protein coupled receptors _ G protein – small heterotrimer (Alpha, beta, gamma), Switch (GDP: off, GTP:on) _ Adenylyl Cyclase – enzyme only activates w/ G(alphas) GTP bound
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• What are the steps of β-Adrenergic Receptor (G proteincoupled receptors) Signal Transduction
– 2nd Messenger – ATP => cAMP – Effector – (great effector molecule) – PKA (protein Kinase A) – Has 4 subunits (2)R & (2) C – If PKA is activated (separated) it can add *P* to other proteins
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What are β-Adrenergic Receptor (G proteincoupled receptors) in Signal Transduction
– Cellular Response – convert glycogen into glucose – Amplification – 1 Epinephrine ~> 20 cAMP – general increase\ – Termination • G protein timer – GTP ~> GDP (end of 2nd messenger) • Phosphodiesterases - Breaks 3’ bond - Phosphatases would remove *P*
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• What is β-Adrenergic Receptor (G protein-coupled receptors) Signal Transduction
– Termination • Desensitization 1. Binding of epinephrine (E) to b-adrenergic receptor triggers dissociation of Gsbg from Gsa 2. Gsbg recruits bARK to the membrane, where it phosphorylates Ser residues at the carboxyl terminus of the receptor. 3. barr binds to the phosphorylated carboxyl-terminal domain of the receptor. 4. Receptor-arrestin complex enters the cell by endocytosis. 5. In endocytic vesicle, arrestin dissociates; receptor is dephosphorylated and returned to cell surface.
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• Regulation of Transcription by Steroid Hormone Receptors in Signal Transduction
– Signal - hormone – Receptor/Effector - receptor – Cellular Response – change in gene express and what that protein does in the cell
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Signal Transduction • Other 2nd Messengers of Signal transduction?
Ca 2+ & cAMP
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How do these 4 signal transduction do all the work?
Different pathways “mix and match” basic components – “cross talk & intergration
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If Adenylyl Cyclase is inhibited what happens?
does not produce cAMP
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Examples of Mix and Match
All G protein coupled (G protein / effector molecule
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• G protein inhibitors
– Cholera Toxin – Gs can not hydrolyze GTP – more active- can’t shut off switch – Pertussis Toxin – Whooping cough – modify Gi – GDP can’t be removed – more signaling to AC
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Signal Transduction • Giα
inhibits cAMP
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