3 Биологические мембраны. Обмен веществом (1160072), страница 15
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Activetransport, by contrast, results in the accumulation of a solute on oneChapter 10 Biological Membranes and Transportside of a membrane. Active transport is thermodynamically unfavorable (endergonic), and occurs only when coupled (directly or indirectly)to an exergonic process such as the absorption of sunlight, an oxidationreaction, the breakdown of ATP, or the concomitant flow of some otherchemical species down its concentration gradient. In primary activetransport, solute accumulation is coupled directly to an exergonic reaction (e.g., conversion of ATP to ADP + Pi). Secondary active transportoccurs when endergonic (uphill) transport of one solute is coupled tothe exergonic (downhill) flow of a different solute that was originallypumped uphill by primary active transport.The amount of energy needed for the transport of a solute against agradient can easily be calculated from the initial concentration gradient. The general equation for the free-energy change in the chemicalprocess that converts S into P isAG = AG°' + RT In [P]/[S](10-1)where R is the gas constant 8.315 J/mol • K and T is the absolute temperature.
When the "reaction" is simply transport of a solute from aregion where its concentration is Ci to another region where its concentration is C2, no bonds are made or broken and the standard freeenergy change, AGO/, equals zero. The free-energy change for transport, AGt, is thenAGt = RT In {C2/CJ(10-2)For a tenfold gradient, the cost of moving 1 mol of an uncharged soluteacross the membrane separating two compartments at 25 °C is thereforeAGt = (8.315 J/mol • KX298 K)(ln 10/1) = 5,705 J/molor 5.7 kJ/mol.
Equation 10-2 holds for all uncharged solutes. When thesolute is an ion, its movement without an accompanying counterionresults in the endergonic separation of positive and negative charges.The energetic cost of moving an ion therefore depends on the differenceof electrical potential across the membrane as well as the difference inthe chemical concentrations (that is, the electrochemical potential):AGt = RT In (Cz/CO + ZJAi//(10-3)where Z is the charge on the ion, J is the Faraday constant (96,480J/V • mol), and Ae/r is the transmembrane electrical potential (in volts).Eukaryotic cells typically have electrical potentials across theirplasma membranes of the order of 0.05 to 0.1 V, so the second term ofEquation 10-3 can be a significant contribution to the total free-energychange for transporting an ion.
Most cells maintain ion gradientslarger than tenfold across their plasma or intracellular membranes,and for many cells and tissues, active transport is therefore a majorenergy-consuming process.The mechanism of active transport is of fundamental importancein biology. As we shall see in Chapter 18, the formation of ATP inmitochondria and chloroplasts occurs by a mechanism that is essentially ATP-driven ion transport operating in reverse. The energy madeavailable by the spontaneous flow of protons across a membrane iscalculable from Equation 10-3; remember that for flow down a concentration gradient, the sign of AG is opposite to that for transport againstthe gradient.287288Part II Structure and CatalysisFigure 10-22 The Na+K+ ATPase is primarily responsible for setting and maintaining the intracellular concentrations of Na+ and K+ and for generating the transmembrane electrical potential, whichit does by moving 3 Na+ out of the cell for every2 K+ it moves in.
The electrical potential is centralto electrical signaling in neurons, and the gradientof Na+ is used to drive uphill cotransport of varioussolutes in a variety of cell types.Transporter binds 3 Na+from the inside of thecell.Phosphorylationfavorsconformation II.Transporterreleases 3 Na+to the outsideand binds 2 K+from the outsideof the cell.Na + (O2K + (d)Dephosphorylationfavorsconformation I.InsideOutsideFigure 10-23 Postulated mechanism of Na+ andK+ transport by the Na+K+ ATPase.
The processbegins with the binding of three Na+ to high-affinity sites on the large subunit of the transport protein on the inner surface of the membrane (a). Thissame part of the large subunit also has the ATPbinding site. Phosphorylation of the transporterchanges its conformation (b) and decreases its affinity for Na+, leading to Na+ release on the outersurface (c). Next, K+ on the outside binds to highaffinity sides on the extracellular portion of thelarge subunit (d), the enzyme is dephosphorylated,reducing its affinity for K+ (e), and K+ is discharged on the inside (f). The transport protein isnow ready for another cycle of Na+ and K+ pumping.3Na+Membrane potential50-70 mV9yNa+K+ ATPase[K + ] = 140 mM[Na + ] = 12 mMExtracellular fluidor blood plasma[K+] = 4 HIM[Na + ] = 145 mMActive Cotransport of Na + and K+ Is Energized by ATPVirtually every animal cell maintains a lower concentration of Na +and a higher concentration of K + than is found in its surrounding medium (in vertebrates, extracellular fluid or the blood plasma) (Fig.10-22).
This imbalance is established and maintained by a primaryactive transport system in the plasma membrane, involving the enzyme Na + K + ATPase, which couples breakdown of ATP to the simultaneous movement of both Na + and K+ against their concentrationgradients.
For each molecule of ATP converted to ADP and Pi? thistransporter moves two K + ions inward and three Na + ions outward,across the plasma membrane. The Na + K + ATPase is an integral membrane protein with two subunits (Mr ~ 50,000 and -110,000), both ofwhich span the membrane.The detailed mechanism by which ATP hydrolysis is coupled totransport remains to be established, but a current working model (Fig.10-23) supposes that the ATPase cycles between two conformations:conformation II, a phosphorylated form (designated P-Enz n ) withhigh affinity for K+ and low affinity for Na + , and conformation I, adephosphorylated form (Enzx) with high affinity for Na + and low affinity for K + .
The conversion of ATP to ADP and Pi occurs in two stepscatalyzed by the enzyme:(1) formation of phosphoenzyme:ATP +ADP + P-Enz n(2) hydrolysis of phosphoenzyme:+ H2Owhich sum to the hydrolysis of ATP: ATP + H2O> ADP + P,.Because three Na + ions move outward for every two K+ ions thatmove inward, the process is electrogenic—it creates a net separationof charge across the membrane, making the inside of the cell negativerelative to the outside.
The resulting transmembrane potential of -50Chapter 10 Biological Membranes and Transportto -70 mV (inside negative relative to outside) is essential to the conduction of action potentials in neurons, and is also characteristic ofmost nonneuronal animal cells.
The activity of this Na + K + ATPase inextruding Na + and accumulating K+ is an essential cell function;about 25% of the energy-yielding metabolism of a human at rest goes tosupport the Na + K + ATPase.Ouabain (pronounced 'wa-ban), a steroid derivative extracted fromthe seeds of an African shrub, is a potent and specific inhibitor of theNa + K + ATPase. Ouabain is a powerful poison used to tip hunting arrows; its name is derived from waba yo, meaning "arrow poison."HOHOH OHOuabainThere Are Three General Types of Transport ATPasesThe Na + K + ATPase is the prototype for a class of transporters (Table10-5), all of which are reversibly phosphorylated as part of the transport cycle—thus the name, P-type ATPase.
All P-type transportATPases share amino acid sequence homology, especially near the Aspresidue that undergoes phosphorylation, and all are sensitive to inhibition by the phosphate analog vanadate. Each is an integral membrane protein having multiple membrane-spanning regions. P-typetransporters are very widely distributed. In higher plants, a P-type H +ATPase pumps protons out of the cell, establishing a difference ofas much as 2 pH units and 250 mV across the plasma membrane.For each proton transported, one ATP is consumed. A similar P-typeATPase is responsible for pumping protons from the bread moldNeurospora, and for pumping H + and K+ across the plasma membranes of cells that line the mammalian stomach, acidifying its contents (Table 10-5).A distinctly different class of transport ATPases is responsible foracidifying intracellular compartments in many organisms. Within thevacuoles of higher plants and of fungi, for example, the pH is maintained well below that of the surrounding cytoplasm by the action ofV-type ATPase-proton pumps.
V-type (for vacuole) ATPases arealso responsible for the acidification of lysosomes, endosomes, theGolgi complex, and secretory vesicles in animal cells. Unlike P-typeATPases, these proton-pumping ATPases (Table 10-5) do not undergocyclic phosphorylation and dephosphorylation, and are not inhibited byvanadate or ouabain.
The mechanism by which they couple ATP hydrolysis to the concentrative transport of protons is not yet known.O"O"Po=v—o~IIOHPhosphateOHVanadate290Part II Structure and CatalysisTable 10-5 Three classes of ion transport ATPasesOrganismType of membraneRole of the ATPaseNa+K+Higher eukaryotesPlasmaH+K+Acid-secreting cells ofmammalsPlasmaMaintains low [Na+], high[K+] inside cell; createstransmembrane electricalpotentialAcidifies contents of stomachH+H+Fungi (Neurospora)Higher plantsPlasmaPlasmaCa2+Higher eukaryotesPlasmaCa2+Muscle cells of animalsSarcoplasmic reticulum(endoplasmic reticulum)AnimalsHigher plantsFungiLysosomal, endosomal,secretory vesiclesVacuolarVacuolarCreates low pH incompartment, activatingproteases and otherhydrolytic enzymesEukaryotesHigher plantsProkaryotesInner mitochondrialThylakoidPlasmaCatalyzes formation of ATPfrom ADP + PjTransported ion(s)P-type ATPasesCreates low pH incompartment; activatingproteases and otherhydrolytic enzymesMaintains low [Ca2+] incytosolSequesters intracellularCa2+, keeping cytosolic[Ca2+] lowV-type ATPasesH+H+F-type ATPasesH+H+H+A third family of ATP-splitting proton pumps plays the central rolein energy-conserving reactions in bacteria, mitochondria, and chloroplasts.
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