Biomedical subjects
C Tanford
Publications and source records attributed to C Tanford.
Overview: mechanism of free energy coupling between ATP hydrolysis and ion transport.
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Sarcoplasmic reticulum calcium pump: a model for Ca2+ binding and Ca2+-coupled phosphorylation.
The conventional alternating access model for Ca2+ transport by the sarcoplasmic reticulum Ca2+ pump is modified, partly on the basis of the proposed MacLennan-Green domain structure for the Ca2+-pump protein. The present model divides the uptake state (E1) of the protein into three substates, differing in the condition of the Ca2+-binding domain. The domain is an open cavity in the first substate and can bind only a single Ca2+ ion. A fast "jaw-closing" (or "hinge-bending") step then partially closes the cavity to generate the second substate that has a second Ca2+-binding site. Occupation of this site is followed by another jaw-closing step that closes the binding cavity and occludes the bound ions. The subsequent translocation step (to form E2) remains unchanged from previous models. The modified model predicts a constant transport stoichiometry of two Ca2+ per pump reaction cycle. It suggests a plausible mechanism for coupling between Ca2+ binding and ATP utilization: the model predicts (in agreement with experiment) that Ca2+ binding should be a mandatory requirement for phosphorylation of the pump protein, though ATP binding per se does not require Ca2+. The model is consistent with high cooperativity in equilibrium binding of Ca2+, both in the absence and presence of ATP.
Amphiphile orientation: physical chemistry and biological function.
The critical role that amphiphiles play in biology is to provide an orienting force, probably the single most important force for structural organization in living matter. It is responsible for the organization of biological lipids into the membranes that define a living cell and prevent mixing of intracellular contents with the inanimate outside world. The same force, much reduced in magnitude, is responsible for the folding of protein polypeptide chains to specific three-dimensional structures, thereby creating the multitude of enzymes of the living cell, and the proteins that traverse the cell membrane for communication between inside and outside. Study of these phenomena has a long and fascinating history, but many important questions--all at the interface between biological and physical chemistry--remain unanswered. What is the mechanism of vesiculation of phospholipid bilayers? What is the functional role of cholesterol in cell membranes? Knowing that molecular clusters are important elements in the structure of liquid water, how do we explain the smooth extrapolation of thermodynamic data from very large to very small hydrophobic interfacial areas?
Circular dichroism of the two major conformational states of mammalian (Na+ + K+)-ATPase.
No alteration in the circular dichroic spectrum of fully active, membrane-bound (Na+ + K+)-ATPase is observed when the protein is cycled between the two major conformational states, E1 and E2. This finding is in agreement with the infrared study by Chetverin and Brazhnikov (J. Biol. Chem. 260 (1985) 7817) and demonstrates that any difference in secondary structure between the two conformers must be less than 2%.
Simple model can explain self-inhibition of red cell anion exchange.
Ion translocation in red cell anion exchange is assumed to occur by means of an alternating access mechanism, in which a critical binding site for the transported ion alternates between two conformational states, each accessible from only one side of the membrane. If this alternating site is located within the transport protein at some distance from one or both surfaces of the membrane, an access channel is required to connect the alternating site to the adjacent bulk solution. This automatically leads to inhibition of transport at high concentrations of the transported ion because release of the ion from the alternating site can occur only via unoccupied channel sites.
Application of the principle of linked functions to ATP-driven ion pumps: kinetics of activation by ATP.
If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct participant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center.
Thermodynamic and kinetic cooperativity in ligand binding to multiple sites on a protein: Ca2+ activation of an ATP-driven Ca pump.
Contrary to common belief, theoretical analysis does not predict any necessary relationship between cooperativity in the equilibrium binding of an ion to multiple binding sites on a protein and cooperativity in the kinetic activation of a reaction for which such binding is prerequisite. The sarcoplasmic reticulum Ca pump protein, for example, has two high-affinity binding sites for Ca2+, here considered to be nearly identical and independent. Equilibrium binding to these sites can be highly cooperative in spite of site-independence, as demonstrated by the well-known allosteric mechanism based on Wyman's principle of linked functions. We show in this paper that kinetic activation of the pump reaction cycle by binding of Ca2+ to these same sites can likewise be a cooperative function of Ca2+ concentration but that the criteria that determine cooperativity in the two situations are different. It is possible to observe kinetic cooperativity without concomitant cooperativity in equilibrium binding and vice versa. Application of these theoretical considerations to experimental data for the pump protein raises questions about the Ca2+ binding mechanism.
Variable stoichiometry in active ion transport: theoretical analysis of physiological consequences.
Active ion transport systems with fixed stoichiometry are subject to a thermodynamic limit on the ion concentration gradients that they can generate and maintain, and their net rates of transport must inevitably decrease as this limit is approached. The capability to vary stoichiometry might thus be physiologically advantageous: a shift to lower stoichiometry (fewer ions pumped per reaction cycle) at increasing thermodynamic load could increase the limit on the supportable concentration gradient and could accelerate the rate of transport under high-load conditions. Here we present a theoretical and numerical analysis of this possibility, using the sarcoplasmic reticulum ATP-driven Ca pump as the example. It is easy to introduce alternate pathways into the reaction cycle for this system to shift the stoichiometry (Ca2+/ATP) from the normal value of 2:1 to 1:1, but it cannot be done without simultaneous generation of a pathway for uncoupled leak of Ca2+ across the membrane. This counteracts the advantageous effect of the change in transport stoichiometry and a physiologically useful rate acceleration cannot be obtained. This result is likely to be generally applicable to most active transport systems.
Incorporation of membrane potential into theoretical analysis of electrogenic ion pumps.
The transport rate of an electrogenic ion pump, and therefore also the current generated by the pump, depends on the potential difference (delta psi) between the two sides of the membrane. This dependence arises from at least three sources: (i) charges carried across the membrane by the transported ions; (ii) protein charges in the ion binding sites that alternate between exposure to (and therefore electrical contact with) the two sides of the membrane; (iii) protein charges or dipoles that move within the domain of the membrane as a result of conformational changes linked to the transport cycle. Quantitative prediction of these separate effects requires presently unavailable molecular information, so that there is great freedom in assigning voltage dependence to individual steps of a transport cycle when one attempts to make theoretical calculations of physiological behavior for an ion pump for which biochemical data (mechanism, rate constants, etc.) are already established. The need to make kinetic behavior consistent with thermodynamic laws, however, limits this freedom, and in most cases two points on a curve of rate versus delta psi will be fixed points independent of how voltage dependence is assigned. Theoretical discussion of these principles is illustrated by reference to ATP-driven Na,K pumps. Physiological data for this system suggest that all three of the possible mechanisms for generating voltage dependence do in fact make significant contributions.
Solubilized monomeric sarcoplasmic reticulum Ca pump protein. Phosphorylation by inorganic phosphate.
Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein. In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca2+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.
Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability.
The method developed previously for formation of unilamellar vesicles from mixed micelles of egg lecithin and octyl glucoside [Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981) Biochemistry 20, 833-840] has been extended to allow for (1) use of nonionic detergents with much lower critical micelle concentrations and (2) variation in the time course of detergent removal. The results demonstrate the importance of kinetic factors, especially in the determination of vesicle size: initially formed vesicles are small, but the size increases slowly thereafter if detergent is not removed too quickly. Vesicle size remains fixed when the molar detergent/lipid ratio falls below about 1/1, and detergent removal becomes increasingly difficult thereafter, presumably because flip-flop of detergent from the inner to the outer leaflet of the bilayer membrane is very slow. Residual detergent (to about 25 mol %) has surprisingly little effect on anion permeability but increases cation permeability to the point where the normal discrimination between anions and cations (in pure lipid vesicles) is lost. Detergent added to initially detergent-free vesicles readily partitions into vesicular membranes (presumably only into the outer leaflet) and has a qualitatively similar effect on permeability. Vesicles produced by this method, regardless of residual detergent level, were found to be predominantly unilamellar: no multilamellar liposomes or other lipid aggregates could be detected within the accuracy of the methods employed.
The sarcoplasmic reticulum calcium pump. Localization of free energy transfer to discrete steps of the reaction cycle.
Free energy transduction in active transport resembles other protein-catalyzed processes, occurring by an ordered sequence of discrete bond-breaking and bond-making steps. The bonds that affect the transported ion directly are chelation bonds, which alter the chemical potential of the bound ion, but not its chemical identity. Available data for the sarcoplasmic reticulum Ca pump (admittedly incomplete) suggest that more than 50% of the free energy transfer may be localized to a single step of the reaction cycle.
Monomeric solubilized sarcoplasmic reticulum Ca pump protein: demonstration of Ca binding and dissociation coupled to ATP hydrolysis.
The sarcoplasmic reticulum Ca-pump protein can be solubilized in monomeric form in nonionic detergents with full retention of ATPase activity. It is impossible to prove directly that coupling between ATP hydrolysis and Ca2+ transport is maintained in the soluble state, because separate compartments for Ca2+ uptake and Ca2+ discharge are required to demonstrate this, but here we provide strong indirect evidence that coupling in fact persists, in both the forward and reverse directions of the normal pump cycle. Demonstration of coupling in the forward direction makes use of the fact that the solubilized protein is structurally labile in the absence of bound Ca2+. Loss of activity accompanying ATP hydrolysis in solution is quantitatively consistent with sequential Ca2+ binding and dissociation during the hydrolysis cycle. Coupling in the reverse direction is demonstrated by the dependence of ATP synthesis on Ca2+ concentration. Although substantial differences between solubilized (monomeric) and membrane-bound protein are shown to exist, as previously reported, they do not affect the main conclusion from this work, which is that the molecular machinery for free-energy coupling in active Ca2+ transport is an inherent property of each individual catalytic polypeptide chain of the pump protein.
Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.
The problem of "mechanism" for the calcium pump may be divided into three parts. (1) It is an enzyme catalyzing the hydrolysis of ATP. (2) At some stage of the reaction cycle it provides a pathway through the otherwise impermeable phospholipid bilayer. (3) The two properties are linked so as to provide exchange of free energy between the two substrates (ATP and Ca2+), without any exchange of matter. The third part is the most interesting, and the mechanistic problem it poses is common to all chemiosmotic free energy transducers. All three aspects of the mechanism are reviewed here, with special emphasis on the remaining experimental questions that need to be resolved. The review will show that even such fundamental questions as the exact stoichiometry of the catalyzed reaction have not yet received definitive answers.
Gel-exclusion chromatography on S1000 Sephacryl: application to phospholipid vesicles.
Sephacryl S1000 has an exclusion diameter of approximately 3000 A and is thus an appropriate gel exclusion medium for size analysis and fractionation of phospholipid vesicles. Calibration is conveniently carried out using polystyrene beads of known diameter eluted with a detergent-containing buffer. Recovery of phospholipid vesicles of different sizes from S1000 presaturated with lipid is greater than 95%, and diameters obtained from a calibration curve using the polystyrene beads are in good agreement with those obtained by negative contrast electron microscopy.
Translocation pathway in the catalysis of active transport.
Possible pathways for translocation across the membrane in active transport processes are examined theoretically. Thermodynamic and kinetic requirements are readily satisfied by an alternating-access mechanism of the kind that has been proposed in the past by several investigators. The essential features of this mechanism (for transport of a single species) are shown to be defined by four explicit conditions. (i) The transport protein must have at least two distinct conformational states, each accessible from only one side of the membrane. (ii) Binding affinity for the transported species is high in the state accessible from the uptake side of the membrane and much lower in the state accessible from the discharge side. (iii) The change from one conformation to the other involves movement of the binding site itself (with the transported species remaining attached) or rearrangement within the site that is topologically equivalent to such movement. (iv) Return to the original conformation occurs with unoccupied binding sites. The analysis demonstrates that a passage through the membrane that is simultaneously accessible from both sides cannot be used for active transport regardless of what the energetics of opening or closing of the passage may be. Even movement from one fixed site to another within the protein, without access to the outside, is virtually excluded as a possible element of the central mechanism. A ligand conduction mechanism for ATP-linked ion transport is in principle conceivable but is subject to restrictions that make it improbable.
Mechanism of free energy coupling in active transport.
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