09/30/2010Biochemistry: Membranes II Membranes II Andy Howard Introductory Biochemistry 30 September 2010

09/30/2010Biochemistry: Membranes II

Membranes II

Andy HowardIntroductory Biochemistry

30 September 2010

09/30/2010 Biochemistry: Membranes II

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Membranes work hard

Transport of various types requires active participation of various proteins and sometimes involves energy input.

Interactions between signaling molecules and receptors occurs at the membrane and allow an external signaling molecule to influence internal behavior.


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What we’ll discuss Membrane transport Review Transporting charges

Pores & Channels Passive Transport

Active Transport Moving large molecules

Signal transduction General Principles

G proteins Adenylyl cyclase Inositol-phospholipid signaling pathway

Receptor tyr kinases


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Cartoons of transport types

From accessexcellence.org


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Thermodynamics ofpassive and active transport• If you think of the transport as a chemical reaction Ain Aout or Aout Ain

• It makes sense that the free energy equation would look like this:

• Gtransport = RTln([Ain]/[Aout])

• More complex with charges;see eqns. 9.4 through 9.6.


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Example Suppose [Aout] = 145 mM, [Ain] = 10 mM,T = body temp = 310K

Gtransport = RT ln[Ain]/[Aout]= 8.325 J mol-1K-1 * 310 K * ln(10/145)= -6.9 kJ mol-1

So the energies involved are moderate compared to ATP hydrolysis


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Charged species Charged species give rise to a factor that looks at charge difference as well as chemical potential (~concentration) difference

Most cells export cations so the inside of the cell is usually negatively charged relative to the outside


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Quantitative treatment of charge differences

Membrane potential (in volts J/coul): = in - out

(there’s an extra in eqn. 9.4) Gibbs free energy associated with difference in electrical potential isGe = zFwhere z is the charge being transported and F is Faraday’s constant, 96485 JV-1mol-1

Faraday’s constant is a fancy name for 1.


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Faraday’s constant Relating energy per moleto energy per coulomb:

Energy per mole of charges,e.g. 1 J mol-1, is1 J / (6.022*1023 charges)

Energy per coulomb, e.g, 1 V = 1 J coul-

1, is1 J / (6.241*1018 charges)

1 V / (J mol-1) =(1/(6.241*1018)) / (1/(6.022*1023) = 96485

So F = 96485 J V-1mol-1


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Total free energy change When charges move, we typically have both a chemical potential difference and an electrical potential difference so

Gtransport = RTln([Ain]/[Aout]) + zF

Sometimes these two effects are opposite in sign, but not always


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Pores and channels

Transmembrane proteins with centralpassage for small molecules,possibly charged, to pass through Bacterial: pore. Usually only weakly selective

Eukaryote: channel. Highly selective. Usually the Gtransport is negative so they don’t require external energy sources

Gated channels: Passage can be switched on Highly selective, e.g. v(K+) >> v(Na+)

Rod MacKinnon


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Gated potassium channels Eukaryotic potassium channels are gated, i.e. they exist in open or closed forms

When open, they allow K+ but not Na+ to pass through based on ionic radius (1.33Å vs. 0.95Å)

Some are voltage gated; others are ligand gated


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Protein-facilitated passive transport All involve negative Gtransport

Uniport: one solute across Symport: two solutes, same direction Antiport: two solutes, opposite directions

Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow

These proteins can be inhibited, reversibly or irreversibly

Diagram courtesySaint-Boniface U.


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Kinetics of passive transport Michaelis-Menten saturation kinetics:

v0 = Vmax[S]out/(Ktr + [S]out) We’ll derive that relationship in the enzymatic case in a later chapter

Vmax is velocity achieved with fully saturated transporter

Ktr is analogous to Michaelis constant:it’s the [S]out value for which half-maximal velocity is achieved.


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Velocity versus [S]out

Transport Velocity

0

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

0.00035

0.0004

0.00045

0.0005

0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045

[S]out

v 0

Vmax = 0.5 mM s-1

Ktr = 0.1 mM


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1/v0 versus 1/[S]outTransport Lineweaver Burk

0

500

1000

1500

2000

2500

3000

3500

4000

4500

-10000 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 10000

1/[S]out, M-1

1/v0, sM-1


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Primary active transport

Energy source is usually ATP or light Energy source directly contributes to overcoming concentration gradient Bacteriorhodopsin: light energy used to drive protons against concentration and charge gradient to enable ATP production

P-glycoprotein: ATP-driven active transport of many nasties out of the cell


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Secondary active transport Active transport of one solute is coupled to passive transport of another

Net energetics is (just barely) favorable

Generally involves antiport Bacterial lactose influx driven by proton efflux

Sodium gradient often used in animals


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Complex case: Na+/K+

pump Typically [Kin] = 140mM, [Kout] = 5mM,[Nain] = 10 mM, [Naout] = 145mM.

ATP-driven transporter:3 Na+ out for 2 K+ inper molecule of ATP hydrolyzed

3Na out: 3*6.9 kJmol-1,2K in: 2*8.6 kJmol-1

= 37.9 kJ mol-1 needed, ~ one ATP

Diagram courtesy

Steve Cook


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What’s this used for? Sodium gets pumped back in in symport with glucose, driving uphill glucose transport

That’s a separate passive transport protein called GluT1

Diagram courtesy

Steve Cook


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How do we transport big molecules? Proteins and other big molecules often internalized or secreted by endocytosis or exocytosis

Special types of lipid vesicles created for transport


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Receptor-mediated endocytosis Bind macromolecule to specific receptor in plasma membrane

Membrane invaginates, forming a vesicle surrounding the bound molecules (still on the outside)

Vesicle fuses with endosome and a lysozome Inside the lysozyome, the foreign material and the receptor get degraded

… or ligand or receptor or both get recycled


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Example: LDL-cholesterol

Diagram courtesyGwen Childs, U.Arkansas for Medical Sciences


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Exocytosis

Materials to be secreted are enclosed in vesicles by the Golgi apparatus

Vesicles fuse with plasma membrane

Contents released into extracellular space

Diagram courtesy LinkPublishing.com


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Transducing signals Plasma membranes contain receptors that allow the cell to respond to chemical stimuli that can’t cross the membrane

Bacteria can detect chemicals:if something useful comes along,a signal is passed from the receptor to the flagella, enabling the bacterium to swim toward the source


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Multicellular signaling

Hormones, neurotransmitters, growth factors all can travel to target cells and produce receptor signals

Diagram courtesy Science Creative Quarterly, U. British Columbia


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Extracellular Signals

Internal behavior ofcells modulated by external influences

Extracellular signals are called first messengers

7-helical transmembrane proteins with characteristic receptor sites on extracellular side are common, but they’re not the only receptors

Image courtesy CSU Channel Islands


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Internal results of signals Intracellular: heterotrimeric G-proteins are the transducers: they receive signal from receptor, hydrolyze GTP, and emit small molecules called second messengers

Second messengers diffuse to target organelle or portion of cytoplasm

Many signals, many receptors, relatively few second messengers

Often there is amplification involved


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Roles of these systems Response to sensory stimuli Response to hormones Response to growth factors Response to some neurotransmitters Metabolite transport Immune response This stuff gets complicated, because the kinds of signals are so varied!


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G proteins Transducers of external signals into the inside of the cell

These are GTPases (GTP GDP + Pi) GTP-bound protein transduces signalsGDP-bound protein doesn’t

Heterotrimeric proteins; association of and subunits with subunit is disrupted by complexation with hormone-receptor complex, allowing departure of GDP & binding of GTP


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G protein cycle Ternary complex

disrupted by binding of receptor complex

G-GTP interacts with effector enzyme

GTP slowly hydrolyzed away

Then G-GDP reassociates with ,

See fig. 9.39 for details

GDP

GTP

GTP

Inactive

Active

GDP

H2O

Pi

Inactive


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Adenylyl cyclase

cAMP and cGMP: second messengers

Adenylyl cyclase converts ATP to cAMP Integral membrane enzyme; active site faces cytosol

cAMP diffuses from membrane surface through cytosol, activates protein kinase A

Protein Kinase A (PKA) phosphorylates ser,thr in target enzymes;action is reversed by specific phosphatases

Cyclic AMP


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Modulators of cAMP

Caffeine, theophylline inhibit cAMP phosphodiesterase, prolonging cAMP’s stimulatory effects on protein kinase A

Hormones that bind to stimulatory receptors activate adenylyl cyclase, raising cAMP levels

Hormones that bind to inhibitory receptors inhibit adenylyl cyclase activity via receptor interaction with the transducer Gi.

O N

N

N

N

O

caffeine

HN

NNO

N

O

theophylline


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Inositol-Phospholipid Signaling Pathway 2 Second messengers derived

from phosphatidylinositol 4,5-bisphosphate (PIP2)

Ligand binds to specific receptor; signal transduced through G protein called Gq

Active form activates phosphoinositide-specific phospholipase C bound to cytoplasmic face of plasma membrane

O

HO

HO

O

OH

OHPO O-

O

O

O

R1

O

O R2

P

O

O-O

PIP2


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PIP2 chemistry Phospholipase C

hydrolyzes PIP2 to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol

Both of these products are second messengers that transmit the signal into the cell

O

OH

HO

O

O

OH

P

O

-OO-

IP3

P O-O

-O

P

O-

OO-

OH

O

O

R1

O

O R2

diacylglycerol


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IP3 and calcium

IP3 diffuses through cytosol and binds to a calcium channel in the membrane of the endoplasmic reticulum

The calcium channel opens, releasing Ca2+ from lumen of ER into cytosol

Ca2+ is a short-lived 2nd messenger too: it activates Ca2+-dependent protein kinases that catalyze phosphorylation of certain proteins

O

OH

HO

O

O

OH

P

O

-OO-

IP3

P O-O

-O

P

O-

OO-


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Calcium homeostasis & IP3

Courtesy Oulu Univ., Finland


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Diacylglycerol and protein kinase C

Diacylglycerol stays @ plasma membrane

Protein kinase C (which exists in equilibrium between soluble & peripheral-membrane form) moves to inner face of membrane; it binds transiently and is activated by diacylglycerol and Ca2+

Protein kinase C catalyzes phosphorylation of several proteins

OH

O

O

R1

O

O R2

diacylglycerol


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Control of inositol-phospholipid pathway After GTP hydrolysis, Gq is inactive so I no longer stimulates Plase C

Activities of 2nd messengers are transient IP3 rapidly hydrolyzed to other things Diacylglycerol is phosphorylated to form phosphatidate


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The big picture

Courtesy bmj.com


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Sphingolipids give rise to 2nd messengers Some signals activate hydrolases that convert sphingomyelin to: sphingosine sphingosine-1-P, and ceramide

Each of these modulates a second messenger

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.


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Interconversions

Courtesy AOCS Lipid Library


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Fates of sphingolipid products Sphingosine inhibits

Protein Kinase C Ceramides activate a protein kinase and a protein phosphatase

Sphingosine-1-P can activate Phospholipase D, which catalyzes hydrolysis of phosphatidylcholine;products are 2nd messengers

Phospholipase DStreptomyceswith phosphatidyl choline boundPDB 2ZE454 kDa monomer2.5Å


p. 44 of 46

Receptor tyrosine kinases

Most growth factors function via a pathway that involves these enzymes

In absence of ligand, 2 nearby tyr kinase molecules are separated

Upon substrate binding they come together, form a dimer

exterior

interior

ligands

Tyr kinase monomers


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Autophosphorylation of the dimer

Enzyme catalyzes phosphorylation of specific tyr residues in the kinase itself; so this is autophosphorylation

Once it’s phosphorylated, it’s activated and can phosphorylate various cytosolic proteins, starting a cascade of events

PP


p. 46 of 46

Insulin receptor

Insulin binds to an 22 tetramer;binding brings subunits together

Each tyr kinase () subunit phosphorylates the other one

The activated tetramer can phosphorylate cytosolic proteins involved in metabolite regulation

Sketch courtesy ofDavidson College, NC

Documents

09/30/2010Biochemistry: Membranes II Membranes II Andy Howard Introductory Biochemistry 30 September 2010