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Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Chapter 10

Membrane Transportto accompany

Biochemistry, 2/e

byReginald Garrett and Charles Grisham

All rights reserved. Requests for permission to make copies of any part of the work

should be mailed to: Permissions Department, Harcourt Brace & Company, 6277Sea Harbor Drive, Orlando, Florida 32887-6777

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Outline

• 10.1 Passive Diffusion

• 10.2 Facilitated Diffusion

• 10.3 Active Transport

• 10.4 - 10.6 Transport Driven by ATP, light, etc.

• 10.7 Group Translocation

• 10.8 Specialized Membrane Pores

• 10.9 Ionophore Antibiotics

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Passive Diffusion

No special proteins needed

• Transported species simply moves

down its concentration gradient - fromhigh [c] to low [c]

• Be able to use Eq. 10.1 and 10.2

•

High permeability coefficients usuallymean that passive diffusion is not the

whole story

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Facilitated Diffusion

G negative, but proteins assist

• Solutes only move in the

thermodynamically favored direction

• But proteins may "facilitate" transport,increasing the rates of transport

• Understand plots in Figure 10.3

• Two important distinguising features: – solute flows only in the favored direction

– transport displays saturation kinetics

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Active Transport Systems

Energy input drives transport

• Some transport must occur such that

solutes flow against thermodynamic

potential• Energy input drives transport

• Energy source and transport machinery

are "coupled" • Energy source may be ATP, light or a

concentration gradient

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The Sodium Pump

aka Na,K-ATPase

• Large protein - 120 kD and 35 kD subunits

• Maintains intracellular Na low and K high• Crucial for all organs, but especially for

neural tissue and the brain

• ATP hydrolysis drives Na out and K in

• Alpha subunit has ten transmembrane

helices with large cytoplasmic domain

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Na,K Transport

• ATP hydrolysis occurs via an E-P

intermediate

• Mechanism involves two enzymeconformations known as E1 and E2

• Cardiac glycosides inhibit by binding to

outside

/ G & G

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Bi h i t 2/ G tt & G i h

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Na,K Transport

• Hypertension involves apparent

inhibition of sodium pump. Inhibition in

cells lining blood• Vessel walls results in Na,Ca

accumulation

• Studies show this inhibitor to beouabain!


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Calcium Transport in Muscle

A process akin to Na,K transport

• Calcium levels in resting muscle cytoplasm are

maintained low by Ca-ATPase - a Ca pump• Calcium is pumped into the sarcoplasmic

reticulum (SR) by a 110 kD protein that is very

similar to the alpha subunit of Na,K-ATPase

• Aspartyl phosphate E-P intermediate is at Asp-

351 and Ca-pump also fits the E1-E2 model


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Biochemistr 2/e Garrett & Grisham

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Biochemistry 2/e Garrett & Grisham

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The Gastric H,K-ATPase

• This is the largest concentration

gradient across a membrane in

eukaryotic organisms!• H,K-ATPase is similar in many respects

to Na,K-ATPase and Ca-ATPase


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Osteoclast Proton PumpsHow your body takes your bones apart!

• Bone material undergoes ongoing remodeling

– osteoclasts tear down bone tissue•

– osteoblasts build it back up

• Osteoclasts function by secreting acid into

the space between the osteoclast membrane

and the bone surface - acid dissolves the Ca-

phosphate matrix of the bone• An ATP-driven proton pump in the membrane

does this!


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The MDR ATPase

aka the P-glycoprotein

• Animal cells have a transport system

that is designed to recognize foreignorganic molecules

• This organic molecule pump recognizes

a broad variety of molecules andtransports them out of the cell using the

hydrolytic energy of ATP


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Light-Driven H + Transport

The Bacteriorhodopsin story • Halobacterium halobium, the salt-loving

bacterium, carries out normal respiration if

O2 and substrates are plentiful

• But when substrates are lacking, it can

survive by using bacteriorhodopsin and

halorhodopsin to capture light energy

• Purple patches of H. halobium are 75% bRand 25% lipid - a "2D crystal" of bR - ideal

for structural studies


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Bacteriorhodopsin

Protein opsin and retinal chromophore

• Retinal is bound to opsin via a Schiff

base link

• The Schiff base (at Lys-216) can be

protonated, and this site is one of the

sites that participate in H+ transport


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Bacteriorhodopsin

• Lys-216 is buried in the middle of the 7-

TMS structure of bR, and retinal lies

mostly parallel to the membrane andbetween the helices

• Light absorption converts all-trans

retinal to 13-cis configuration - seeFigure 10.22


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Bacteriorhodopsin

The protons visit the aspartates....

• Asp-85 and Asp-96 lie on opposite

sides of a membrane-spanning helix

• These remarkable aspartates have pKa

values around 11! (Why?)

•

Protons are driven from Asp-96 to theSchiff base at Lys-216 to Asp-85 and

out of the cell


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Halorhodopsin

• Halorhodopsin transports Cl - instead of H +

• Halorhodopsin has Lys-242 Schiff base but

no aspartates and no deprotonation ofSchiff base during the transport cycle


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y &



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y



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y


Secondary Active Transport

Transport processes driven by ion

gradients

• Many amino acids and sugars areaccumulated by cells in transport

processes driven by ion gradients


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y


Secondary Active Transport

• Symport - ion and the amino acid or

sugar are transported in the same

direction across the membrane

• Antiport - ion and transported species

move in opposite directions

•

Several examples are described inTable 10.2


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y


Group Translocation

The phosphotransferase system (PTS)

• Discovered by Saul Roseman in 1964

• Sugars are phosphorylated from PEP

during transport into E. coli cells

• Four proteins required: EI, HPr, EII, and

EIII


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y



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y


Group Translocation

• EI and HPr are universal and work for

all sugars

• EII and EIII are specific for each sugar• Mechanism involves transfer of P from

PEP to EI and then to HPr and then to 2

sites on EIII and then finallyphosphorylation of sugar


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Porins

Found both in Gram-negative bacteria and inmitochondrial outer membrane

• Porins are pore-forming proteins - 30-50 kD

• General or specific - exclusion limits 600-6000• Most arrange in membrane as trimers

• High homology between various porins

• Porin from Rhodobacter capsulatus has 16-stranded beta barrel that traverses the

membrane to form the pore (with eyelet!)


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Why Beta Sheets?

for membrane proteins??• Genetic economy

• Alpha helix requires 21-25 residues per

transmembrane strand• Beta-strand requires only 9-11 residues

per transmembrane strand

•Thus, with beta strands , a given amountof genetic material can make a larger

number of trans-membrane segments


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The Pore-Forming Toxins

• Lethal molecules produced by many

organisms

• They insert themselves into the host cellplasma membrane

• They kill by collapsing ion gradients,

facilitating entry by toxic agents, orintroducing a harmful catalytic activity


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Colicins

• Produced by E. coli

• Inhibit growth of other bacteria (even

other strains of E. coli )• Single colicin molecule can kill a host!

• Three domains: translocation (T),

receptor-binding (R), and channel-forming (C)


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Clues to Channel Formation

• C-domain: 10-helix bundle, with H8 and

H9 forming a hydrophobic hairpin

• Other helices amphipathic (Fig. 10.30)• H8 and H9 insert, with others splayed

on the membrane surface

• A transmembrane potential causes theamphipathic helices to insert!


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Other Pore-Forming Toxins

• Delta endotoxin also possesses a helix-

bundle and may work the same way

• There are other mechanisms at work inother toxins

• Hemolysin from Staphylococcus aureus

forms a symmetrical pore• Aerolysin may form a heptameric pore -

with each monomer providing 3 beta

strands to a membrane-spanning barrel


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Amphiphilic Helices

form Transmembrane Ion Channels • Many natural peptides form oligomeric

transmembrane channels

• The peptides form amphiphilic -helices• Aggregates of these helices form

channels that have a hydrophobic

surface and a polar center• Melittin (bee venom), magainins (frogs)

and cecropin (from cecropia moths) are

examples


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Amphipathic Helices

• Melittin - bee venom toxin - 26 residues

• Cecropin A - cecropia moths - 37

residues• Magainin 2 amide - frogs - 23 residues

• See Figure 10.35 to appreciate helical

wheel presentation of the amphipathichelix


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The Magainin Peptides

• Discovered by Michael Zasloff

• He noticed that incisions on Xenopus

laevis (African clawed frog) healedwithout infection, even in bacteria-filled

aquarium water

• He deduced that the frogs produced asubstance that protected them from

infection!


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The Cecropins

• Produced by Hyalophora cecropia (the

cecropia moth - see Figure 10.36)

• Induced when the moth is challenged bybacterial infections

• These peptides are thought to form -

helical aggregates in membranes,creating an ion channel in the center of

the aggregate


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Gap Junctions

Vital connections for animal cells

• Provide metabolic connections

• Provide a means of chemical transfer

• Provide a means of communication

• Permit large number of cells to act in

synchrony


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Gap Junctions

• Hexameric arrays of a single 32 kD

protein

• Subunits are tilted with respect tocentral axis

• Pore in center can be opened or closed

by the tilting of the subunits, e.g. asresponse to stress


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Ionophore AntibioticsMobile carrier or pore (channel)

• How to distinguish? Temperature!

• Pores will not be greatly affected by

temperature, so transport rates are

approximately constant over largetemperature ranges

• Carriers depend on the fluidity of the

membrane, so transport rates are highlysensitive to temperature, especially near the

phase transition of the membrane lipids


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Valinomycin A classic mobile carrier

• A depsipeptide - a molecule with both

peptide and ester bonds

• Valinomycin is a dodecadepsipeptide

• The structure places several carbonyl

oxygens in the center of the ring structure

• Potassium and other ions coordinate the

oxygens• Valinomycin-potassium complex diffuses

freely and rapid across membranes


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Gramicidin A classic channel ionophore

• Linear 15-residue peptide - alternating D & L

• Structure in organic solvents is double

helical• Structure in water is end-to-end helical

dimer

•

Unusual helix - 6.3 residues per turn with acentral hole - 0.4 nm or 4 A diameter

• Ions migrate through the central pore


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biochem 10

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