Mechanism of free energy coupling in active transport.
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Adenosine Diphosphate↗
Biomedical subjects
C Tanford
Publications and source records attributed to C Tanford.
Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations.
Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.
Binding Sites↗
Steady state of an ATP-driven calcium pump: limitations on kinetic and thermodynamic parameters.
A numerical analysis was carried out to explore limitations on kinetic and thermodynamic parameters for an ATP-driven Ca pump. A conventional pump reaction cycle was employed, with a transport stoichiometry of two Ca ions per cycle. Rigid requirements were imposed to represent the needs of physiological function, defined as the ability to maintain the cytoplasmic Ca concentration below 10(-7) M against a 3 mM concentration on the opposite side of the membrane. Realistic physical limits were placed on the magnitudes of rate constants for individual reaction steps. Reversibility under laboratory conditions was assumed. The results show that these requirements can be satisfied simultaneously only if the equilibrium constant for binding Ca from the cytoplasmic (uptake) side of the membrane is much larger than the binding constant on the discharge side. More generally, the results demonstrate that limitations on rate constants make it possible for the pump to maintain an adequate rate only if steady-state levels of kinetically important (slowly reacting) reaction intermediates do not become too disparate. Experimental data for the sarcoplasmic reticulum calcium pump support these theoretical conclusions.
Adenosine Triphosphate↗
Mechanism of active transport: free energy dissipation and free energy transduction.
The thermodynamic pathway for "chemiosmotic" free energy transduction in active transport is discussed with an ATP-driven Ca2+ pump as an illustrative example. Two innovations are made in the analysis. (i) Free energy dissipated as heat is rigorously excluded from overall free energy bookkeeping by focusing on the dynamic equilibrium state of the chemiosmotic process. (ii) Separate chemical potential terms for free energy donor and transported ions are used to keep track of the thermodynamic state of each substrate through the reaction cycle. These procedures clarify the mechanism of free energy transduction, even without step-by-step analysis. The results show that free energy exchange must occur in its entirety among protein-bound species. Imposition of conditions for an adequate rate of physiological function further indicates (i) that the standard free energy of hydrolysis of protein-bound ATP (to yield protein-bound products) needs to differ substantially from the standard free energy of hydrolysis in solution and (ii) that binding sites for the transported ions must have different affinities when facing opposite sides of the membrane. The results also demonstrate that step-by-step "basic" free energy changes (often used in the form of free energy level diagrams) are inherently unsuited for analysis of the mechanism of free energy transduction.
Adenosine Triphosphate↗
Simple model for the chemical potential change of a transported ion in active transport.
The mechanism for active transport of ions across a membrane probably involves two distinct conformational states of the transport protein, in which the binding sites for the transported ion face opposite sides of the membrane. It is likely that the binding affinity for the ion changes in synchrony with the change in site orientation, such that the affinity is high on the uptake side of the membrane and low on the discharge side. A structural model is proposed for the transmembrane portion of such a protein, based on the known multihelical structure of bacteriorhodopsin. This structure is well adapted to a cyclical alternation between two conformations that differ simultaneously in orientation and binding affinity. No unfolding of the helices or other significant alterations in secondary structure is required. The model is explicitly intended as a hypothetical representation of the E1 and E2 states of ATP-driven Na+,K+ and Ca2+ pumps.
Allosteric Regulation↗
Effects of detergent micelles on the recombination reaction of opsin and 11-cis-retinal.
When detergent-solubilized proteins interact with hydrophobic or amphiphilic molecules in the presence of detergent micelles, the solubility of the latter species in the micelles must be included in both thermodynamic and kinetic treatments. In this paper, we derive equations which describe the distribution of species present at equilibrium for a system in which a detergent-solubilized protein binds a hydrophobic (or amphiphilic) ligand. We have applied the formalism developed in this paper to the reaction describing the formation of rhodopsin from its apoprotein and 11-cis-retinal. Qualitatively, the results demonstrate that a significant portion of the observed decrease in the extent of recombination for rhodopsin solubilized in either sodium cholate or Tween 80 may be attributed to the partition of retinal into detergent micelles and that a detergent-induced protein denaturation need not be invoked to explain the data. We also discuss results for rhodopsin solubilized in a nonionic detergent (octaethylene glycol n-dodecyl ether) in which the detergent is clearly causing irreversible loss of the capability to recombine with 11-cis-retinal.
Detergents↗
Different states of aggregation for unbleached and bleached rhodopsin after isolation in two different detergents.
Phospholipid-free rhodopsin has been purified in the detergents sodium cholate and octaethylene glycol n-dodecyl ether (C12E8). In both detergents, the native absorption spectrum of the unbleached protein is maintained; however, upon photolysis, the preparation in C12E8 loses its ability to recombine with 11-cis-retinal, whereas the preparation in cholate does not. The circular dichroic spectra of the protein in the two detergents are nearly identical, indicating that the secondary structure of the protein is the same in the two detergents. The state of association of the protein in the two detergents is different. In sodium cholate, the smallest species present was found to be a trimer of the rhodopsin polypeptide chain, and this association was unaffected by exposure to light. On the other hand, in C12E8, the protein is monomeric and undergoes a nonspecific aggregation process on exposure to light. These results suggest that protein--protein interactions may play an important role in the stabilization of the native structure of rhodopsin.
Animals↗
Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: van't Hoff analysis of enthalpy changes.
The magnesium-dependent phosphorylation of sarcoplasmic reticulum (Ca2+)-AtPase by inorganic phosphate (Mg2+ + Pi + E = Mg.E-P) was studied in purified leaky AtPase vesicles as a function of temperature (20-30 degrees C). A bireactant scheme was used to determine equilibrium constants, and the corresponding enthalpy changes ( delta H degrees vh) were determined by van't Hoff analysis. At all temperatures, the binding of Pi and Mg(2+) to the enzyme was synergistic. The equilibrium constants showed only a modest temperature dependence, with delta H degrees vh varying from 3 to 13 kcal/mol. In particular, the delta H degrees vh Mg(2+) binding to the unoccupied enzyme was 3 +/ 2 kcal/mol. These data contrast with a recent calorimetric study under comparable conditions (Epstein, M., Kuriki, Y., Biltonen, R., & Racker, E. (1980) Biochemistry 17, 5564) which reported no significant binding synergism and a delta H degrees cal for Mg(2+) binding of -76 kcal/mol. A possible reason for the discrepancy between calorimetric and van't Hoff enthalpy determinations is given. In agreement with other previous work, the overall reaction was found to be accompanied by a positive entropy change.
Animals↗
Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.
Removal of detergent from mixed micelles of egg yolk phosphatidylcholine and octyl glucoside leads to formation of unilamellar phospholipid vesicles with a diameter of about 230 nm. The same procedure applied to mixed micelles containing the transmembrane protein glycophorin A, in addition to lipid and detergent, produces vesicles of the same size with glycophorin incorporated into the bilayer. The pure lipid vesicles are highly impermeable to both anions and cations, and incorporation of up to 220 molecules of glycophorin per vesicle has little effect on permeability.
Animals↗
Chemical potential of bound ligand, an important parameter for free energy transduction.
The chemical potential (mu(L,b)) of a ligand L bound to a protein or enzyme can be rigorously defined, and this paper describes some of its properties in relation to other thermodynamic parameters, with emphasis on thermodynamic parameters that may be used in the elucidation of the mechanism of biological free energy transduction. Free energy transduction involves the transfer of free energy from one molecule to another, and the actual transfer may often occur while both molecules are bound to the transducer enzyme, which means that mu(L,b) for one bound ligand increases at the expense of mu(L,b) for the other. The free energy change for the overall reaction may be very small, and it is not possible to express the phenomenon of transfer, in thermodynamic terms, without the explicit use of mu(L,b) as a parameter.
Journal Article↗
Proton and hydroxide ion permeability of phospholipid vesicles.
The apparent permeability of H+ through phospholipid bilayers was determined by measuring H+ efflux from large unilamellar phospholipid vesicles with internal space buffered at pH 4. The value obtained is about 10(-9) cm/sec at room temperature, five orders of magnitude lower than was recently reported for the combined permeability for H+ and OH- [Nichols, J. W. & Deamer, D. W. (1980) Proc. Natl. Acad. Sci. USA, 77, 2038-2042]. The apparent permeability measured in this way is the sum of contributions from the movement of H+ and of uncharged species (HCl or HNO3) in equilibrium with anions in the solution. There is evidence that the uncharged species make the dominant contribution and that the permeability coefficient for H+ per se is no larger than 5 X 10(-12) cm/sec. An attempt to measure OH- permeability by use of vesicles buffered at pH 10 did not give a conclusive result because the vesicle walls appeared to be damaged by exposure to this pH. An apparent permeability coefficient of about 10(-7) cm/sec was estimated for undamaged membranes.
Acids↗
Equilibrium state of ATP-driven ion pumps in relation to physiological ion concentration gradients.
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Adenosine Triphosphate↗
Hydrostatic pressure in small phospholipid vesicles.
The internal solvent-filled cavity of a singlewalled spherical phospholipid vesicle must be at essentially the same pressure as the aqueous medium outside the vesicle. Whether or not the bilayer itself is under elevated pressure cannot at present be determined.
Journal Article↗
Interfacial free energy and the hydrophobic effect.
Interfacial free energies demonstrate clearly that the antipathy between hydrocarbon and water rests on the strong attraction of water for itself. However, the unfavorable free energy associated with this antipathy, per unit area of contact between bulk hydrocarbon and water, is about 3-fold larger than a similar figure derived from solubility data per unit area of contact between a single dissolved hydrocarbon molecule and water. The discrepancy illustrates the difficulty in applying macroscopic concepts such as "interfacial surface" at the molecular level and can be formally resolved, at least qualitatively, by the predicted effect of surface curvature on surface tension.
Journal Article↗
Denaturation of the tryptic fragments of the calcium (II) adenosine triphosphatase from sarcoplasmic reticulum by guanidinium hydrochloride.
Primary and secondary fragments of the Ca2+-adenosine triphosphatase from sarcoplasmic reticulum are resistant to complete denaturation by guanidinium hydrochloride, a property characteristic of many intrinsic membrane proteins. None of the fragments display a single cooperative transition from ordered structure to random coil suggesting each fragment contains several domains of differing resistance to guanidinium hydrochloride denaturation. The data suggest that the native enzyme has at least three membrane-embedded domains, with an externally accessible link between each.
Alkylation↗
Behavior of fragmented calcium (II) adenosine triphosphatase from sarcoplasmic reticulum in detergent solution.
The behavior of Ca2+-ATPase from sarcoplasmic reticulum in detergent solution was compared with that of Ca2+-ATPase which had been cleaved in half by limited trypsin digestion. Attempts to dissociate the fragments (I and II) with an excess of detergent micelles demonstrated that fragments I and II are structurally dependent upon each other, and that they must be denatured in order to be dissociated. Partial dissociation of the fragmented ATPase was found to occur in the bile salt detergents, deoxycholate and cholate, and optical data showed that there was an accompanying change in conformation. No dissociation of the fragmented ATPase was observed in nonionic detergents. The fragmented ATPase retained the same specific activity and stability as the intact ATPase under a variety of conditions when solubilized in Tween 80 or dodecyl octaoxyethylene glycol monoether. The data demonstrate that the noncovalent interactions that maintain the native conformation of the ATPase are not affected by either trypsin cleavage or solubilization in nonionic detergent solution.
Animals↗
Association of the major coat protein of fd bacteriophage with phospholipid vesicles.
The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar "membrane-bound" conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly beta-type conformation in detergent solution, maintained a beta conformation when associated with a vesicle phospholipid bilayer.
Amino Acid Sequence↗