- Email: [email protected]
[19] Modulation of membrane protein function by surface potential
[19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
387
The lipid coumarin is a probe of interfacial polarity as emphasized previously 4,6 and as illustrated recently for a glyeosurfactant, u Finally, it should be noted that the coumarin may be used not only as an analytical tool for probing surface potentials but as a synthetic tool for the design of sensors probing interfacial effects induced by agonists, enzymes, or cells, with direct transduction of the fluorescence signal to a semiconductor. 35 Acknowledgments We thank the Deutsche Forsehungsgemeinsehafland the Fonds der Chemisehen Industile for their generoussupport. 34C. J. Drummond, G. G. Warr, F. Grieser, B. W. Ninham, and D. F. Evans, J. Phys. Chem. 89, 2103 (1985). 35p. Fromherz and W. Arden, J. Am. Chem. Soc. 102, 6211 (1980).
[ 19] Modulation of M e m b r a n e Protein Function by Surface Potential By REINHARD K.R~MER
Introduction Nearly all biomembranes bear a net negative surface charge which gives rise to a negative surface potential. This will affect the concentration of cations and anions in the region adjacent to the membrane and may thus influence kinetic parameters of enzymes and carriers located in the membrane. In a few cases the influence of surface charge and surface potential on transport activity has been analyzed in situ, i.e., with membrane proteins in their natural surroundings, e.g., in bacteria, ~ in mitochondria, 3'4 and in chloroplasts. 5 Furthermore, experiments have been reported where the surface charge was artificially changed within the natural membrane, for instance by adding charged surfactants to mitochondria. ~'7 G. W. F. H. Borst-Pauwels, Biochim. Biophys. Acta 650, 88 (1981). 2 A. P. R. Theuvenet and G . W . F . H . Borst-Panwels, Biochim. Biophys. Acta 734, 62 (1983). 3 H. Rottenberg, Mod. Cell Biol. 4, 47 (1985). 4 I. M. Meller, C. J. Kay, and J. M. Palmer, Biochem. J. 223, 761 (1984). s j. Barber, Annu. Rev. Plant Physiol. 33, 261 (1982). 6 j. Duszynski and L. Woitczak, FEBSLett. 40, 72 (1974). 7 L. Woitezak, FEBSLett. 44, 25 (1974).
METHODS IN ENZYMOLOGY, VOL. 171
Copyright© 1989by AcademicPress,Inc. All rightsofreprodnctionin any formreserved.
388
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[19]
However, in all these cases severe complications arise due to the high protein content of the membrane and due to the possibility of an asymmetrical charge distribution. This has eventually led to the reduction of experimental complexity, i.e., to the use of reconstituted systems. Under the less complex conditions prevailing in the reconstituted system, the influence of surface potential on membrane-bound enzymes has been extensively analyzed, s,9 In these studies, membrane-bound enzymes catalyzing reactions with charged substrates were influenced by the surface potential, which modulated the apparent Km values but did not change the Vm~. This is in agreement with the simple assumption that variation in the surface charge density changes the concentration of charged substrates at the active site of the enzyme close to the surface of the membrane. In the case of carrier proteins the situation is more complicated. It is not only the diffusion step of charged substrates from the surrounding bulk phase to the binding site that may be influenced by surface charges, but also essential conformational change(s) of the carrier/substrate complex during the reaction cycle of the carder protein. Additionally, it has to be remembered that carders, in contrast to membrane-bound enzymes, are necessarily influenced by surface charges at both of the two different membrane sides. This is even more significant when considering that biological membranes are in general asymmetric with respect to their phospholipid and charge distribution, l° The reconstituted A D P - A T P carrier from the inner mitochondrial membrane provides an ideal tool for studying all the factors of the basic question m t h e influence of surface charges and surface potential on the carrier function--for three main reasons: (1) the adenine nucleotide carder transports the highly negatively charged substrates, ADP and ATP. (2) The natural surroundings of this membrane protein, the inner mitochondrial membrane, contain a high amount of negatively charged phospholipids, especially cardiolipin. (3) The inner mitochondrial membrane is asymmetric with respect to the distribution of surface charges.l° These facts show clearly that the reconstituted A D P - A T P carrier is an ideal model system for analyzing the modulating influence of surface charge and surface potential on the kinetic parameters of membrane-bound enzymes and transport systems. s L. Woitczakand M. J. Nalecz,Fur. J. Biochem. 94, 99 (1979). 9 M. 3. Nalecz, J. Zborowski,K. S. Famulsld, and L. Woitczak,Eur. (1980). to j. A. F. Op den Kamp,Annu. Rev. Biochem. 48, 47 (1979).
J. Biochem.
112, 75
[ 19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
389
Experimental The purpose of this chapter is not only to describe how carrier function can be modulated by surface potential, but to correlate quantitatively a certain surface potential to its effect on definite kinetic parameters of membrane-bound enzymes or carriers. Thus, apart from the preparation of suitable reconstituted proteoliposomes forming the basis for the following experiments, both the appropriate determination of surface potential or surface charge and the analysis of the characteristic kinetic constants of the enzymatic or transport process will be described.
Preparation of Proteoliposomes For this type of experiment it is necessary to use a definite lipid composition. However, this prerequisite is in many cases not easy to fulfill. The phospholipids commonly used for physicochemical studies of membrane surface potential, e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with saturated fatty acids, do not usually provide a suitable membrane environment for studying the function of carrier proteins. Highly unsaturated phospholipids that are in general necessary for the activity of these membrane proteins are both expensive and difficult to handle. We generally use purified egg yolk phospholipids ~t for pilot studies, since this material has a very low content of negatively charged lipids. The surface charge of liposomes prepared from this material prove to be reproducibly low and easy to determine, t2 For more elaborate experiments we isolate pure phosphatidylcholine and phosphatidylethanolamine from egg yolks by column chromatography. ~3 In contrast to many preparations of commercially available phospholipids from natural sources, liposomes prepared from this material prove to be nearly completely free from net negative charges at the surface. ~2 Liposomes can be prepared by a number of different methods~4; however, for the experiments discussed here, we are limited by the necessity to incorporate functionally active proteins into these membranes. ~s On the one hand, we are restricted to certain detergent removal techniques, such as dialysis, ~5 column procedures, ~4 and removal by hydrophobic absorption, ~4,~6 or, on the other hand, to the freeze/thaw/sonication techml M. A. Wells and D. J. Hanahan, this series, Vol. 14, p. 178. 12 R. Krimer, Biochim. Biophys. Acta 735, 145 (1983). 13 G. B. Ansell and J. N. Hawthorne, "Phospholipids." Elsevier, Amsterdam, 1964. 14F. Szoka, Jr. and D. Papahadjopoulos, Annu. Rev. Biophys. Bioeng. 9, 467 (1980). 1~ E. Racker, this series, Vol. 55, p. 699. 16 M. Ueno, C. Tanford, and J. A. Reynolds, Biochemistry 23, 3070 (1984).
390
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[19]
nique/7,~s The reconstitution method for the model system described here, i.e., the A D P - A T P carrier from mitochondria, has already been described in detail. 19 Variation in the surface charge of the reconstituted proteoliposomes can be achieved in two ways. First, charged lipids can be included in the lipid mixture before the preparation of proteoliposomes, leading to a more or less uniform distribution of the charged molecules in the bilayer of the proteoliposomes. This is the method of choice if negatively charged phospholipids, e.g., phosphatidylserine or cardiolipin, are used to establish negative surface potentials. The second method for changing the surface charge consists of an addition of lipophilic molecules to the preformed proteoliposomes, leading to an asymmetric distribution of the charged substances located mainly at the outside of the bilayer. This method is preferentially used with low-molecular-weight substances such as dicetyl phosphate, free fatty acids (negatively charged), or alkyltrimethylammonium bromide (positively charged). These compounds can be added either as micellar solution in aqueous buffers, or dissolved in small amounts of organic solvent; their effect on the reconstituted liposomes must be tested separately. The rate of flip-flop of these molecules to the inner half of the bilayer of the liposomes can easily be tested by measurement of surface potential. ~2 At this point there is an important fact to consider regarding the methods of variation of surface potential in natural membranes. This variation is usually achieved by changing the ionic strength in the surroundings of the (negatively) charged biomembrane. It has to be kept in mind that this procedure leads only to a variation of the actual surface potential in the phase adjacent to the membrane, which thus modulates the concentration of charged ligands. The intrinsic surface charge density of the membrane which may directly influence the inserted membrane proteins is in general not changed by this method (if we neglect direct binding to the surface charges), since usually the concentration of monovalent ions is varied. However, a will be seen in the following, it is in fact a variation in the surface charge density that may be significant for the elucidation of the influences on particular functions of the membrane protein. In order to measure significant effects of surface potential on membrane proteins in the case of a prevailing low density of surface charges, i.e., less than 10% negatively charged lipids, one has to reduce the ionic strength outside the liposomes to relatively low levels. This is sometimes rather ~7M. Kasahara and P. Hinkle, J. Biol. Chem. 252, 7384 (1977). ~s R. Kr~rner and M. Klingenberg, FEBS Lett. 82, 363 (1977). ~9R. Kr~lmer, this series, Vol. 125, p. 610.
[19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
391
difficult to achieve, since it is in many cases not possible to prepare the proteoliposomes in low-salt buffers. Especially in the case of exchange carriers, the appropriate activating ions and substrates have to be present in the interior phase of the liposomes in sufficiently high concentrations and thus have to be added from the start. For the separation of the proteoliposomes from the surrounding ions ultracentrifugation is usually not a convenient method, due to the insufficient sedimentation behavior of liposomes. Our experience is that prolonged dialysis of proteoliposomes in low-salt buffer in general leads to aggregation of the vesicles and to inactivation of the reconstituted protein. We prefer to use gel filtration on Sephadex G-75 (fine) in buffers composed mainly of sucrose or mannitol, the concentrations of which balance the internal osmolarity. The volume of the sample applied to the column has to be relatively high, i.e., it has to amount to at least 10% of the bed volume of the column, in order to prevent peak broadening and tailing due to the high viscosity of the proteoliposome dispersion.
Measurement of Surface Potential Many methods for measuring surface potential have been described.2° Since membrane proteins and especially carder proteins in proteoliposomes are not of unlimited stability, the method here should be both fast and not harmful to the incorporated proteins. We have found two methods suitable for this purpose. The use of 2-p-toluidinylnaphthalene 6-sulfate (TNS) as charged fluorescent distribution probe is convenient for determining the surface potential of proteoliposomes. This method has been excellently described in the literature. 2~-23 Although it leads to a possible underestimation of the true surface potential, ~2a° it proved to be the most accurate method of those applicable in the minute time range. In order to avoid artifacts and to increase the reliability of this method, we routinely determine the surface potential of a particular proteoliposome preparation several times, varying the ionic strength (monovalent ions). Then we calculate the corresponding surface charge density which gives rise to the surface potentials observed under the varying ion concentrations. 12,2°,~ Only when the calculated surface charge is found to be constant over a considerable range of ionic strength values can the measurement be accepted. 2o D. Cafiso, A. McLaughlin, S. MeLaughlin, and A. Winiski, this volume [16]. 2J S. McLaughlin, Curt. Top. Membr. Transp. 9, 1977. 22 M. Eisenberg, T. Oresalfi, T. Riccio, and S. McLaughlin, Biochemistry 18, 5213 (1979). 23 H. Rottenberg, this volume [17]. 24 R. Kramer and G. Kiirzinger, Biochim. Biophys. Acta 765, 353 (1984).
392
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[ 19]
The second method we use for the determination of surface potential is based on the pK shift of the lipophilic pH indicator alkylumbelliferone. This method was developed by Fromherz25 and provides a very accurate measurement of surface potential. For the determination we add the dye to the preformed proteoliposomes, thereby monitoring the surface potential of the outer half of the membrane.12 The measurement of surface potential by alkylumbelliferone is more complicated than the use of the fluorescent distribution probe TNS, since it requires a simultaneous determination of fluorescence and pH. Furthermore, it cannot be applied to highly negatively charged membranes, due to the strong pK shift into the alkaline region that results under these circumstances. This method is, however, not only more accurate than the determinations using distribution probes, but also less prone to artifacts caused by adsorption of the probe to proteins.
Determination of Kinetic Constants The kinetic analysis of the activity of the reconstituted membrane protein has to be carded out using the same preparation of proteoliposomes as that used for the determination of surface potential, since the reproducibility of exactly the same reconstitution conditions is in general not very high. The most obvious parameter modulated by surface potential is the apparent dissociation constant, Kd, of a charged ligand dissociating from its protein target in the neighborhood of the membrane surface. Classically, according to the Boltzmann equation, a change in the K~ of an ion with the charge z can be described by the equation
K6 ~pp-- Kd exp( zqg/a/k T) where q is the electronic charge.~2.26 The influence of surface potential on the apparent K,, of a membranebound enzyme or on the transport affinity (Km or Kt) of a carrier protein is in general much more complex. An excellent theoretical description of this complex situation has been published recently.27 In some cases, in which the influence of surface charges on the apparent Km has been quantitatively analyzed, linear reciprocal plots (i.e., Miehaelis-Menten kinetics) and a clear correlation between surface potential and kinetic properties have been found, in spite of these difficulties. In other words, a relationship is found that is similar to that expressed in the above equation for the 25 p. Fromherz, this volume [18]. 26 E. Katchalski, I. Silman, and R. Goldman, Adv. Enzyraol. 34, 445 (1971). 27 p. W. J. A. Baxts and G. W. F. H. Borst-Pauwels, Biochirn. Biophys. Acta 1113, 51 (1985).
[19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
393
dependence of the apparent Kd on surface potential. These favorable circumstances held true not only for membrane-bound enzymes incorporated into liposomes,8,9 but also, for instance, for NADH dehydrogenase in native plant mitochondria4 and for the reconstituted A D P - A T P carrier from the inner mitochondrial membrane. ~2 It is important to consider a further possible effect of the surface potential on carrier kinetics. A change in the surface potential may introduce a simultaneous change in the difference of the potential between the two membrane interfaces, i.e., the membrane potential3 ~ Especially when electrogenic carriers are involved, this may lead to additional complexity in the experimental situation. Thus, in order to simplify the analysis of surface potential effects on carrier kinetics, it is usually of benefit to cancel out an influence of the membrane potential by using symmetric membranes or by providing an equivalent flux of monovalent cations, e.g., K +, through the membrane. Although only the relatively simple conditions of a reconstituted system permit a thorough study of the more or less complete spectrum of modulating parameters with respect to the influence of surface charge, the problem of protein orientation arises in the experiments with reconstituted transmembranous proteins. In some cases, the orientation of the incorporated protein is easy to determine, due to the accessibility of its substrates only from one side of the membrane. In general, however, the protein orientation has to be determined for every reconstitution procedure. In the case of the adenine nucleotide carrier, for instance, this can be achieved by the use of the side-specific inhibitors carboxyatractylate and bongkrekate.2s Especially in experiments where surface charges have been introduced only at the outer side of the reconstituted proteoliposomes by adding charged lipophilic compounds to the preformed vesicles (see above), the orientation of the membrane protein has to be considered. The influence of surface potential on the enzyme or transport activity, i.e., the Vm~x,should also always be analyzed, although in principle this may not seem to be very obvious (see below). Again, the reconstituted A D P - A T P cartier provides a good example where the influence on Vm~ can be clearly discriminated from a Km effect? TM
Correlation of Kinetic Parameters with Surface Charge To make a quantitative correlation of various kinetic parameters with the observed surface potential, one must always be aware of the complexity of possible interactions and influences at certain steps within a complete reaction cycle of an enzyme or carrier system. As mentioned before, the 2s R. Kr~imer and M. Klingenberg,
Biochemistry18, 4209
(1979).
394
MODEL MEMBRANES A N D THEIR CHARACTERISTICS
[ 19]
most basic and simple approach is that of determining the dependence of pure Kd values on a variation of surface potential. When a real transport function is investigated, one can in general discriminate between influences of surface potential on affinity parameters (transport affinity Km) and those on velocity parameters ( V ~ ) . In these cases we have to take into account that the complex equation for the apparent transport affinity may also include rate constants (velocity parameters) and that the equation for V ~ may include dissociation constants (affinity parameters), as shown in numerous analysis of transport kinetics. 29-31 However, at least a qualitative but nevertheless important statement can be made. If the charged membrane surface predominantly modulates the affinity parameters of the particular membrane protein, one can attribute this to a simple surface potential effect. The most convincing example of this situation is achieved by modulating the kinetic properties of membrane-bound enzymes, s'9 where no effect at all on V ~ is found. If, on the other hand, a definite effect on the V ~ ' of the protein function is observed, one can correlate this with an influence of the surface charge. In other words, a direct interaction of the charged lipid molecule at the membrane surface with the membrane protein under investigation can be assumed in the latter case, in contrast to the unspecific and indirect effect of the surface potential generated by these surface charges.
29R. J. Turner, I Membr. Biol. 76, 1 (1983). 3oD. Sanders. U.-P. Hansen, D. Gradmann, and C. L. Slayman, J. Membr. Biol. 77, 123 (1974). 3~ R. K~mer and M. Klingenberg, Biochemistry21, 1082 (1982).
MODULATION OF PROTEIN BY SURFACE POTENTIAL
387
The lipid coumarin is a probe of interfacial polarity as emphasized previously 4,6 and as illustrated recently for a glyeosurfactant, u Finally, it should be noted that the coumarin may be used not only as an analytical tool for probing surface potentials but as a synthetic tool for the design of sensors probing interfacial effects induced by agonists, enzymes, or cells, with direct transduction of the fluorescence signal to a semiconductor. 35 Acknowledgments We thank the Deutsche Forsehungsgemeinsehafland the Fonds der Chemisehen Industile for their generoussupport. 34C. J. Drummond, G. G. Warr, F. Grieser, B. W. Ninham, and D. F. Evans, J. Phys. Chem. 89, 2103 (1985). 35p. Fromherz and W. Arden, J. Am. Chem. Soc. 102, 6211 (1980).
[ 19] Modulation of M e m b r a n e Protein Function by Surface Potential By REINHARD K.R~MER
Introduction Nearly all biomembranes bear a net negative surface charge which gives rise to a negative surface potential. This will affect the concentration of cations and anions in the region adjacent to the membrane and may thus influence kinetic parameters of enzymes and carriers located in the membrane. In a few cases the influence of surface charge and surface potential on transport activity has been analyzed in situ, i.e., with membrane proteins in their natural surroundings, e.g., in bacteria, ~ in mitochondria, 3'4 and in chloroplasts. 5 Furthermore, experiments have been reported where the surface charge was artificially changed within the natural membrane, for instance by adding charged surfactants to mitochondria. ~'7 G. W. F. H. Borst-Pauwels, Biochim. Biophys. Acta 650, 88 (1981). 2 A. P. R. Theuvenet and G . W . F . H . Borst-Panwels, Biochim. Biophys. Acta 734, 62 (1983). 3 H. Rottenberg, Mod. Cell Biol. 4, 47 (1985). 4 I. M. Meller, C. J. Kay, and J. M. Palmer, Biochem. J. 223, 761 (1984). s j. Barber, Annu. Rev. Plant Physiol. 33, 261 (1982). 6 j. Duszynski and L. Woitczak, FEBSLett. 40, 72 (1974). 7 L. Woitezak, FEBSLett. 44, 25 (1974).
METHODS IN ENZYMOLOGY, VOL. 171
Copyright© 1989by AcademicPress,Inc. All rightsofreprodnctionin any formreserved.
388
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[19]
However, in all these cases severe complications arise due to the high protein content of the membrane and due to the possibility of an asymmetrical charge distribution. This has eventually led to the reduction of experimental complexity, i.e., to the use of reconstituted systems. Under the less complex conditions prevailing in the reconstituted system, the influence of surface potential on membrane-bound enzymes has been extensively analyzed, s,9 In these studies, membrane-bound enzymes catalyzing reactions with charged substrates were influenced by the surface potential, which modulated the apparent Km values but did not change the Vm~. This is in agreement with the simple assumption that variation in the surface charge density changes the concentration of charged substrates at the active site of the enzyme close to the surface of the membrane. In the case of carrier proteins the situation is more complicated. It is not only the diffusion step of charged substrates from the surrounding bulk phase to the binding site that may be influenced by surface charges, but also essential conformational change(s) of the carrier/substrate complex during the reaction cycle of the carder protein. Additionally, it has to be remembered that carders, in contrast to membrane-bound enzymes, are necessarily influenced by surface charges at both of the two different membrane sides. This is even more significant when considering that biological membranes are in general asymmetric with respect to their phospholipid and charge distribution, l° The reconstituted A D P - A T P carrier from the inner mitochondrial membrane provides an ideal tool for studying all the factors of the basic question m t h e influence of surface charges and surface potential on the carrier function--for three main reasons: (1) the adenine nucleotide carder transports the highly negatively charged substrates, ADP and ATP. (2) The natural surroundings of this membrane protein, the inner mitochondrial membrane, contain a high amount of negatively charged phospholipids, especially cardiolipin. (3) The inner mitochondrial membrane is asymmetric with respect to the distribution of surface charges.l° These facts show clearly that the reconstituted A D P - A T P carrier is an ideal model system for analyzing the modulating influence of surface charge and surface potential on the kinetic parameters of membrane-bound enzymes and transport systems. s L. Woitczakand M. J. Nalecz,Fur. J. Biochem. 94, 99 (1979). 9 M. 3. Nalecz, J. Zborowski,K. S. Famulsld, and L. Woitczak,Eur. (1980). to j. A. F. Op den Kamp,Annu. Rev. Biochem. 48, 47 (1979).
J. Biochem.
112, 75
[ 19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
389
Experimental The purpose of this chapter is not only to describe how carrier function can be modulated by surface potential, but to correlate quantitatively a certain surface potential to its effect on definite kinetic parameters of membrane-bound enzymes or carriers. Thus, apart from the preparation of suitable reconstituted proteoliposomes forming the basis for the following experiments, both the appropriate determination of surface potential or surface charge and the analysis of the characteristic kinetic constants of the enzymatic or transport process will be described.
Preparation of Proteoliposomes For this type of experiment it is necessary to use a definite lipid composition. However, this prerequisite is in many cases not easy to fulfill. The phospholipids commonly used for physicochemical studies of membrane surface potential, e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with saturated fatty acids, do not usually provide a suitable membrane environment for studying the function of carrier proteins. Highly unsaturated phospholipids that are in general necessary for the activity of these membrane proteins are both expensive and difficult to handle. We generally use purified egg yolk phospholipids ~t for pilot studies, since this material has a very low content of negatively charged lipids. The surface charge of liposomes prepared from this material prove to be reproducibly low and easy to determine, t2 For more elaborate experiments we isolate pure phosphatidylcholine and phosphatidylethanolamine from egg yolks by column chromatography. ~3 In contrast to many preparations of commercially available phospholipids from natural sources, liposomes prepared from this material prove to be nearly completely free from net negative charges at the surface. ~2 Liposomes can be prepared by a number of different methods~4; however, for the experiments discussed here, we are limited by the necessity to incorporate functionally active proteins into these membranes. ~s On the one hand, we are restricted to certain detergent removal techniques, such as dialysis, ~5 column procedures, ~4 and removal by hydrophobic absorption, ~4,~6 or, on the other hand, to the freeze/thaw/sonication techml M. A. Wells and D. J. Hanahan, this series, Vol. 14, p. 178. 12 R. Krimer, Biochim. Biophys. Acta 735, 145 (1983). 13 G. B. Ansell and J. N. Hawthorne, "Phospholipids." Elsevier, Amsterdam, 1964. 14F. Szoka, Jr. and D. Papahadjopoulos, Annu. Rev. Biophys. Bioeng. 9, 467 (1980). 1~ E. Racker, this series, Vol. 55, p. 699. 16 M. Ueno, C. Tanford, and J. A. Reynolds, Biochemistry 23, 3070 (1984).
390
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[19]
nique/7,~s The reconstitution method for the model system described here, i.e., the A D P - A T P carrier from mitochondria, has already been described in detail. 19 Variation in the surface charge of the reconstituted proteoliposomes can be achieved in two ways. First, charged lipids can be included in the lipid mixture before the preparation of proteoliposomes, leading to a more or less uniform distribution of the charged molecules in the bilayer of the proteoliposomes. This is the method of choice if negatively charged phospholipids, e.g., phosphatidylserine or cardiolipin, are used to establish negative surface potentials. The second method for changing the surface charge consists of an addition of lipophilic molecules to the preformed proteoliposomes, leading to an asymmetric distribution of the charged substances located mainly at the outside of the bilayer. This method is preferentially used with low-molecular-weight substances such as dicetyl phosphate, free fatty acids (negatively charged), or alkyltrimethylammonium bromide (positively charged). These compounds can be added either as micellar solution in aqueous buffers, or dissolved in small amounts of organic solvent; their effect on the reconstituted liposomes must be tested separately. The rate of flip-flop of these molecules to the inner half of the bilayer of the liposomes can easily be tested by measurement of surface potential. ~2 At this point there is an important fact to consider regarding the methods of variation of surface potential in natural membranes. This variation is usually achieved by changing the ionic strength in the surroundings of the (negatively) charged biomembrane. It has to be kept in mind that this procedure leads only to a variation of the actual surface potential in the phase adjacent to the membrane, which thus modulates the concentration of charged ligands. The intrinsic surface charge density of the membrane which may directly influence the inserted membrane proteins is in general not changed by this method (if we neglect direct binding to the surface charges), since usually the concentration of monovalent ions is varied. However, a will be seen in the following, it is in fact a variation in the surface charge density that may be significant for the elucidation of the influences on particular functions of the membrane protein. In order to measure significant effects of surface potential on membrane proteins in the case of a prevailing low density of surface charges, i.e., less than 10% negatively charged lipids, one has to reduce the ionic strength outside the liposomes to relatively low levels. This is sometimes rather ~7M. Kasahara and P. Hinkle, J. Biol. Chem. 252, 7384 (1977). ~s R. Kr~rner and M. Klingenberg, FEBS Lett. 82, 363 (1977). ~9R. Kr~lmer, this series, Vol. 125, p. 610.
[19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
391
difficult to achieve, since it is in many cases not possible to prepare the proteoliposomes in low-salt buffers. Especially in the case of exchange carriers, the appropriate activating ions and substrates have to be present in the interior phase of the liposomes in sufficiently high concentrations and thus have to be added from the start. For the separation of the proteoliposomes from the surrounding ions ultracentrifugation is usually not a convenient method, due to the insufficient sedimentation behavior of liposomes. Our experience is that prolonged dialysis of proteoliposomes in low-salt buffer in general leads to aggregation of the vesicles and to inactivation of the reconstituted protein. We prefer to use gel filtration on Sephadex G-75 (fine) in buffers composed mainly of sucrose or mannitol, the concentrations of which balance the internal osmolarity. The volume of the sample applied to the column has to be relatively high, i.e., it has to amount to at least 10% of the bed volume of the column, in order to prevent peak broadening and tailing due to the high viscosity of the proteoliposome dispersion.
Measurement of Surface Potential Many methods for measuring surface potential have been described.2° Since membrane proteins and especially carder proteins in proteoliposomes are not of unlimited stability, the method here should be both fast and not harmful to the incorporated proteins. We have found two methods suitable for this purpose. The use of 2-p-toluidinylnaphthalene 6-sulfate (TNS) as charged fluorescent distribution probe is convenient for determining the surface potential of proteoliposomes. This method has been excellently described in the literature. 2~-23 Although it leads to a possible underestimation of the true surface potential, ~2a° it proved to be the most accurate method of those applicable in the minute time range. In order to avoid artifacts and to increase the reliability of this method, we routinely determine the surface potential of a particular proteoliposome preparation several times, varying the ionic strength (monovalent ions). Then we calculate the corresponding surface charge density which gives rise to the surface potentials observed under the varying ion concentrations. 12,2°,~ Only when the calculated surface charge is found to be constant over a considerable range of ionic strength values can the measurement be accepted. 2o D. Cafiso, A. McLaughlin, S. MeLaughlin, and A. Winiski, this volume [16]. 2J S. McLaughlin, Curt. Top. Membr. Transp. 9, 1977. 22 M. Eisenberg, T. Oresalfi, T. Riccio, and S. McLaughlin, Biochemistry 18, 5213 (1979). 23 H. Rottenberg, this volume [17]. 24 R. Kramer and G. Kiirzinger, Biochim. Biophys. Acta 765, 353 (1984).
392
MODEL MEMBRANES AND THEIR CHARACTERISTICS
[ 19]
The second method we use for the determination of surface potential is based on the pK shift of the lipophilic pH indicator alkylumbelliferone. This method was developed by Fromherz25 and provides a very accurate measurement of surface potential. For the determination we add the dye to the preformed proteoliposomes, thereby monitoring the surface potential of the outer half of the membrane.12 The measurement of surface potential by alkylumbelliferone is more complicated than the use of the fluorescent distribution probe TNS, since it requires a simultaneous determination of fluorescence and pH. Furthermore, it cannot be applied to highly negatively charged membranes, due to the strong pK shift into the alkaline region that results under these circumstances. This method is, however, not only more accurate than the determinations using distribution probes, but also less prone to artifacts caused by adsorption of the probe to proteins.
Determination of Kinetic Constants The kinetic analysis of the activity of the reconstituted membrane protein has to be carded out using the same preparation of proteoliposomes as that used for the determination of surface potential, since the reproducibility of exactly the same reconstitution conditions is in general not very high. The most obvious parameter modulated by surface potential is the apparent dissociation constant, Kd, of a charged ligand dissociating from its protein target in the neighborhood of the membrane surface. Classically, according to the Boltzmann equation, a change in the K~ of an ion with the charge z can be described by the equation
K6 ~pp-- Kd exp( zqg/a/k T) where q is the electronic charge.~2.26 The influence of surface potential on the apparent K,, of a membranebound enzyme or on the transport affinity (Km or Kt) of a carrier protein is in general much more complex. An excellent theoretical description of this complex situation has been published recently.27 In some cases, in which the influence of surface charges on the apparent Km has been quantitatively analyzed, linear reciprocal plots (i.e., Miehaelis-Menten kinetics) and a clear correlation between surface potential and kinetic properties have been found, in spite of these difficulties. In other words, a relationship is found that is similar to that expressed in the above equation for the 25 p. Fromherz, this volume [18]. 26 E. Katchalski, I. Silman, and R. Goldman, Adv. Enzyraol. 34, 445 (1971). 27 p. W. J. A. Baxts and G. W. F. H. Borst-Pauwels, Biochirn. Biophys. Acta 1113, 51 (1985).
[19]
MODULATION OF PROTEIN BY SURFACE POTENTIAL
393
dependence of the apparent Kd on surface potential. These favorable circumstances held true not only for membrane-bound enzymes incorporated into liposomes,8,9 but also, for instance, for NADH dehydrogenase in native plant mitochondria4 and for the reconstituted A D P - A T P carrier from the inner mitochondrial membrane. ~2 It is important to consider a further possible effect of the surface potential on carrier kinetics. A change in the surface potential may introduce a simultaneous change in the difference of the potential between the two membrane interfaces, i.e., the membrane potential3 ~ Especially when electrogenic carriers are involved, this may lead to additional complexity in the experimental situation. Thus, in order to simplify the analysis of surface potential effects on carrier kinetics, it is usually of benefit to cancel out an influence of the membrane potential by using symmetric membranes or by providing an equivalent flux of monovalent cations, e.g., K +, through the membrane. Although only the relatively simple conditions of a reconstituted system permit a thorough study of the more or less complete spectrum of modulating parameters with respect to the influence of surface charge, the problem of protein orientation arises in the experiments with reconstituted transmembranous proteins. In some cases, the orientation of the incorporated protein is easy to determine, due to the accessibility of its substrates only from one side of the membrane. In general, however, the protein orientation has to be determined for every reconstitution procedure. In the case of the adenine nucleotide carrier, for instance, this can be achieved by the use of the side-specific inhibitors carboxyatractylate and bongkrekate.2s Especially in experiments where surface charges have been introduced only at the outer side of the reconstituted proteoliposomes by adding charged lipophilic compounds to the preformed vesicles (see above), the orientation of the membrane protein has to be considered. The influence of surface potential on the enzyme or transport activity, i.e., the Vm~x,should also always be analyzed, although in principle this may not seem to be very obvious (see below). Again, the reconstituted A D P - A T P cartier provides a good example where the influence on Vm~ can be clearly discriminated from a Km effect? TM
Correlation of Kinetic Parameters with Surface Charge To make a quantitative correlation of various kinetic parameters with the observed surface potential, one must always be aware of the complexity of possible interactions and influences at certain steps within a complete reaction cycle of an enzyme or carrier system. As mentioned before, the 2s R. Kr~imer and M. Klingenberg,
Biochemistry18, 4209
(1979).
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MODEL MEMBRANES A N D THEIR CHARACTERISTICS
[ 19]
most basic and simple approach is that of determining the dependence of pure Kd values on a variation of surface potential. When a real transport function is investigated, one can in general discriminate between influences of surface potential on affinity parameters (transport affinity Km) and those on velocity parameters ( V ~ ) . In these cases we have to take into account that the complex equation for the apparent transport affinity may also include rate constants (velocity parameters) and that the equation for V ~ may include dissociation constants (affinity parameters), as shown in numerous analysis of transport kinetics. 29-31 However, at least a qualitative but nevertheless important statement can be made. If the charged membrane surface predominantly modulates the affinity parameters of the particular membrane protein, one can attribute this to a simple surface potential effect. The most convincing example of this situation is achieved by modulating the kinetic properties of membrane-bound enzymes, s'9 where no effect at all on V ~ is found. If, on the other hand, a definite effect on the V ~ ' of the protein function is observed, one can correlate this with an influence of the surface charge. In other words, a direct interaction of the charged lipid molecule at the membrane surface with the membrane protein under investigation can be assumed in the latter case, in contrast to the unspecific and indirect effect of the surface potential generated by these surface charges.
29R. J. Turner, I Membr. Biol. 76, 1 (1983). 3oD. Sanders. U.-P. Hansen, D. Gradmann, and C. L. Slayman, J. Membr. Biol. 77, 123 (1974). 3~ R. K~mer and M. Klingenberg, Biochemistry21, 1082 (1982).