Proc. Nati. Acad. Sci. USA Vol. 73, No. 7, pp. 2280-2284, July 1976 Biochemistry
Antibody evidence for different conformational states of ADP,ATP translocator protein isolated from mitochondria (membranes/transport/protein conformation)
BOB B. BUCHANAN*, W. EIERMANN, P. RICCIOt, H. AQUILA, AND M. KLINGENBERGt Institute of Physiological Chemistry and Physical Biochemistry, University of Munich, Munich, Federal Republic of Germany
Communicated by Daniel I. Arnon, April 20,1976
stages in the transport of ADP or ATP because a transition between the m-state and the c-state occurs only during the translocation of one molecule of ADP or ATP. The knowledge of the existence of the translocator protein in two different orientation states was used to develop a procedure for the solubilization and purification of the translocator protein. The protein was first isolated after stabilization against denaturation by the formation of the CAT-protein complex (6-9), and was more recently isolated as the somewhat less stable BKA-protein complex (P. Riccio and M. Klingenberg, unpublished data). New evidence will be presented for two conformational states of the ADP,ATP translocator protein, based on the use of specific antibodies against the isolated ligand-protein complexes. The antibody against the CAT-protein complex characteristic of the c-state did not react with the translocator protein in the m-state. Conversely, the antibody against the BKA-protein characteristic of the m-state did not react with the protein in the c-state.
Consistent with the previously proposed reABSTRACT orientation mechanism for the ADPATP transl ator protein of mitochondria, evidence has now been obtained for the existence of two distinct conformational states of the isolated translocator protein. Previous studies indicated that when the mitochondrial translocator protein is in the c-state (i.e., when its binding site faces the cytosol side) the protein binds primarily the ligand carboxyatractylate (CAT), and when the translocator protein is in the m-state (i.e., when its binding site faces the mitochondrial matrix) the translocator protein binds primarily bongkrekate. Direct evidence for this formulation has now come from the application of antibodies to the isolated translocator
protein-ligand complex. Two antibodies were produced against the ADPATP translocator protein isolated from beef heart mitochondria. One antibody, which was produced against the protein isolated as the CAT-binding protein complex, was found to be highly specific for that complex and did not react with the protein in the conformation state conferred by the bongkrekate ligand. This antibody did not cover the CAT-bindingsite, as evidenced by the exchange of unlabeled CAT with [ SICAT bound to the translocator protein. However, the same antibody inhibited a transition of the protein from the c-state to the m-state, as evidenced by an inhibition of the displacement of [5S CAT by bofigkrekate (added jointly with ADP). It appears, therefore, that the antibody immobilized the translocator protein in the
METHODS Protein Purification. Methods for purification from beef heart mitochondria of the CAT-binding protein in Triton X-100 solutions were as described previously (7, 8). BKAbinding protein was solubilized from mitochondria loaded with ADP + BKA and purified by a similar procedure. The properties of the BKA-protein preparation will be reported elsewhere. Hydroxylapatite Fractions of Preloaded Mitochondria. Hydroxylapatite fractions containing the ADP,ATP translocator were obtained from "preloaded" mitochondria prepared by adding to 1 ml of thawed beef heart mitochondria (10 mg of protein) 40MgM of each of the following: (1) CAT; (2) ADP; (3) BKA; (4) ADP; and (5) ADP. Numbers (1) and (2) were incubated for 7 min without further additions; the following addition (to 40 MM) was made after 2 min to the remaining samples: (3) ADP; (4) BKA; and (5) CAT. Incubation of (3)-(5) was continued for an additional 5 min, and all samples were then centrifuged at 9000 X g for 15 min. The supernatant fraction was discarded and the translocator was solubilized by suspending the pellet in 1 ml of a solution (pH 7.2) containing 4% (vol/vol) Triton X-100, 20 mM 4-morpholinepropanesulfonic acid, and 0.5 M NaCl. After 1 hr, samples were centrifuged (1 hr at 143,000 X g), the pellet was discarded, and the supernatant fractions were applied to hydroxylapatite columns (0.8 X 12.5 cm) equilibrated beforehand with a solution (pH 7.2) containing 0.5% Triton X-100, 10 mM morpholinepropanesulfonic acid, and 100 mM NaCl. The translocator was then eluted with the same buffer solution. The translocator peak fractions (determined by sodium dodecyl sulfate gel electrophoresis) were combined and concentrated by ultrafiltration. Antibodies., For antibody production, rabbits received
c-state.
The second antibody produced against the (somewhat less pure) ADPATP translocator protein, isolated as the bongkrekate-binding protein complex, did not react with the CATbinding protein. Thus, the second antibody appeared to be specific for the translocator protein in the m-state. Neither antibody inhibited mitochondrial ADPATP transport. One feature of membranes central to their importance in living cells is their ability to catalyze a highly ordered transport of metabolites and ions by specific translocator proteins. In most eukaryotic cells, a key intracellular transport process is catalyzed by the ADP,ATP translocator protein (cf. ref. 1). Research in the last several years has considerably clarified the mode of action of the ADP,ATP translocator protein. It has been possible to demonstrate that the ADP,ATP binding site of the translocator protein is oriented either to the outer (cytosol) or to the inner (mitochondrial matrix) face of the membrane (2-5). Each of these states of protein orientation may be stabilized by the binding of a specific high-affinity ligand: carboxyatractylate (CAT) for the cytosol or c-state and bongkrekate (BKA) for the matrix or m-state. These two states appear to reflect different Abbreviations: CAT, carboxyatractylate; BKA, bongkrekate. * Permanent address: Department of Cell Physiology, University of California, Berkeley, Calif. 94720. t Present address: Istituto di Chimica Biologica, Universiti de Bari, Via Amendola 165A, 70126 Bari, Italy. t To whom reprint requests should be addressed: c/o Institut fur Physiologische Chemie und Physikalische Biochemie der Universitit Munchen, 8 Mfinchen 2, Goethestrasse 33, Federal Republic of Germany.
2280
Biochemistry:
Buchanan et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
.6
2281
CAT-PROT., BHM
'4
CATQ0
Ec 12 0 0
Tco
-.
1.0
'a 0.8
CAT+BSA
Antibody
C)
0
0
0o 0o
CAT-PROT RLM CAT-PROT NCM
CAT+DPG.BSA
o 0.4 Anti body (0.5% Triton)
0.2 --.A6...
o
20
30
40
50
60
70
80
90
M1 rGIbt~n FIG. 1. Precipitation of beef heart mitochondrial CAT-protein by its antibody. Antibody or control gamma globulin (69 mg/ml) was added stepwise to a 1 ml cuvette (1 cm light path) containing 0.6 ml of 56 mM sodium barbital buffer supplemented with 56 mM sodium acetate (pH 8.4) and 98;tg of CAT-protein that was dissolved in 0.2 ml of a solution containing 20 mM morpholinepropanesulfonic acid buffer, 50 mM NaCl, and 0.5% Triton X-100 (pH 7.2). Additional Triton X-100 (0.5%) was added to the indicated sample. Optical density was measured at 600 nm. Time between gamma globulin additions was 45 min. Length of experiment was 14 hr.
through their rear footpads the CAT-translocator complex in solution (pH 7.2) containing 5 mM morpholinepropanesulfonic acid, 50 mM NaCl, and the small amount of Triton X-100 that remained after extraction of the original 5% Triton solution three times with diethyl ether. The BKA-translocator complex was injected into rabbits in a solution containing 5% Triton X-100. Prior to injection, an emulsion of incomplete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) was added to the protein solution in a volume ratio of 3:1 (10). The initial injection of 2 mg of protein was followed after 3 days by a booster injection of 1 mg of protein and by four subsequent booster injections, each of 1 mg of protein, administered at weekly intervals. Antibody was detected in the serum by the Ouchterlony double-diffusion technique after 1 month. The serum was then collected biweekly over a 2-week period, after which the rabbits were killed and bled for collection of total serum. The gamma globulin fraction was isolated from the pooled sera of each rabbit by ammonium sulfate fractionation and DEAE-cellulose chromatography (11). Control serum collected from the same animals prior to injection was fractionated in the same manner. Other Methods. Methods for sodium dodecyl sulfate gel electrophoresis, preparation of [35S]CAT, and for estimation of protein were as described previously (3). a
RESULTS Prior to its solubilization and purification for the experiments reported below, the ADP,ATP translocator was fixed in either of its two orientations in the membrane by binding to it one of the high-affinity ligands, CAT or BKA (2, 3). Because of their very high affinity for the translocator (Kd < 10-10 M), these ligands stabilize the translocator in a specific orientation to a much greater extent than does ADP or ATP. After binding CAT, the binding site of the translocator faces the outer (cytosol) surface of the membrane ("c-state"); after binding BKA, the binding site faces the inner (matrix) surface of the membrane ("m-state") (6). The orientations conferred by these ligands appear to be retained during the procedures used for the solubilization and purification of the translocator.
FIG. 2. Specificity of antibody against the beef heart mitochondrial CAT-protein in Ouchterlony double diffusion. The wells and precipitin band are shown in a high-contrast negative of the Ouchterlony plate. Center well, 5 1d of CAT-protein antibody (AB) (37 mg/ml). Outer wells, 5 ,d of one of the following: CAT-protein (2 mg/ml) from beef heart mitochondria (BHM); partly purified CATprotein (2 mg/ml) from rat liver mitochondria (RLM); CAT-protein (2 mg/ml) from Neurospora crassa mitochondria (NCM); or the equivalent amount of free CAT (40MuM), or CAT (40 MM) + bovine serum albumin (BSA) (2 mg/ml), or CAT (40 MM) + cardiolipin (DPG) (0.03 mg/ml) + BSA (2 mg/ml). The only Triton X-100 present was that added to the outer wells with the 5 ,l samples of the CATprotein or to the other agents tested for antigenic activity. Each of these solutions contained 0.5% Triton X-100.
Antibody against the CAT-translocator complex Fig. 1 demonstrates the presence of an antibody against the purified ADP,ATP translocator that was fixed in the c-state by the high-affinity ligand CAT (6). The presence of the antibody was demonstrated by its ability to precipitate specifically the CAT-translocator complex (henceforth called the CAT-protein). Precipitation was sensitive to the detergent Triton X-100 and was nearly completely inhibited by the 0.5% concentration routinely used to maintain the CAT-protein in solution. The inhibition by Triton X-100 was found in other experiments to be due not to interference of binding of the antibody to the translocator but to inhibition of precipitate formation after binding had occurred. In these experiments, the free antibody that remained after incubation with the CAT-protein dissolved in different concentrations of Triton X-100 was estimated by its capacity to precipitate a second aliquot of the CAT-protein in Ouchterlony double diffusion (cf. Fig. 2). To determine its specificity, the antibody against the CATprotein from beef heart mitochondria was tested for its ability to precipitate other proteins. As shown in Fig. 2, the antibody reacted only with its specific antigen when tested by the Ouchterlony double-diffusion technique. (Positive reaction is indicated by the presence of a precipitin line.) There was no reaction of antibody with fresh preparations of the pure CAT-protein from Neurospora crassa mitochondria or with the partially purified counterpart from rat liver mitochondria. Also, CAT, alone or in the presence of bovine serum albumin or cardiolipin, gave no precipitin reaction. The inability of antibodies against the CAT-protein from beef heart mitochondria to react with the CAT-protein from other sources or with CAT bound to bovine serum albumin indicates that the translocator protein serves as the antigenic determinant in antibody formation, independently of the bound CAT. Further evidence that CAT itself does not elicit antibody formation (i.e., function as a hapten group) was provided by the findings that the CAT-protein antibody did not bind [a5S]CAT in equilibrium dialysis and that added CAT had no effect on precipitation of the CAT-protein in Ouchterlony double-diffusion experiments. The specificity of the antibody for a particular conformation of the translocator was shown in a comparison of its reactivity
2282
Biochemistry: Buchanan et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
UNLOADED
BKA-PROT BHM
0
ADP-BKA
0
CAT-LOADED
v
ADP
BKA-ADP
FIG. 3. Reaction of CAT-protein antibody with the translocator from mitochondria preloaded with different ligands. Center well, 5 !d of CAT-protein antibody (69 mg/ml). Outer wells, 5 ,d of one of the following: translocator preparations (hydroxylapatite column passthrough fractions) that were preloaded as indicated with CAT, ADP, BKA + ADP, ADP + BKA, or ADP + CAT. The column passthrough fractions contained approximately equal amounts of the translocator as determined by sodium dodecyl sulfate gel electrophoresis. Other conditions were as in Fig. 2.
with preparations obtained from beef heart mitochondria preloaded with different ligands. Fig. 3 shows that, of several different ligands (including BKA), the antibody precipitated only the translocator preparation derived from mitochondria preloaded with CAT. Other experiments revealed that the antibody did not react with the derivatives of the CAT-protein obtained after treatment with sodium dodecyl sulfate or ATP (unpublished data). We conclude, therefore, that the antibodies are specific not only for the particular translocator native to beef heart mitochondria but also for a specific conformation of that translocator that is conferred by CAT. The conferral of a specific conformation to the ADP,ATP translocator on binding CAT is also substantiated by other properties of the isolated protein: in contrast to the nonliganded protein, the CAT-protein in Triton X-100 solution is (i) not adsorbed strongly on hydroxylapatite, and (ii) maintained in a state largely protected against denaturation and subsequent degradation by proteinases (P. Riccio and M. Klingenberg, unpublished data). The BKA-translocator complex appears to be considerably less stabilized in its conformation. Antibody against the BKA-translocator complex Because the translocator would be expected, on the basis of earlier findings, to become fixed in a different, well-defined conformation on binding BKA (m-state) (5, 9), we attempted to obtain an antibody against the purified BKA-translocator complex (henceforth called the BKA-protein). The isolated BKA-protein complex appears to contain the same polypeptide chain as the CAT protein (P. Riccio and M. Klingenberg, unpublished results) and therefore, in agreement with the postulates of the reorientation mechanism, can be assumed to represent another conformation of the same protein. Fig. 4 shows not only that antibodies were produced against the BKA-protein, but also that they were quite specific: a precipitin reaction was observed only with the antigen and not with the CAT-protein or its sodium dodecyl sulfate derivative. In no case was there a reaction between the BKA-protein or the CATprotein and the control gamma globulin fraction obtained with rabbits prior to immunization. Effect of antibodies on change in translocator conformation As sensitive indicators of conformation, the antibodies against the translocator might be expected to influence the conformational changes that accompany transition of the protein between the c- and m-states. It is known from earlier work that
Sodim dodecyl s heated CAT-PROT. CAT- PROT NCM
0
0
0
00 0
CAT- PROT BHM
BKA+BSA
BKA+DPG+BSA FIG. 4. Specificity of antibody against the beef heart mitochondrial BKA-protein. Center well, 5 ,g of BKA-protein antibody (40 mg/ml). Outer wells, 5 ,g of one of the following: BKA-protein from beef heart mitochondria (BHM) (1.2 mg/ml); CAT-protein from beef heart mitochondria (1.5 mg/ml); BKA (50 AsM) + cardiolipin (DPG) (0.03 mg/ml) + bovine serum albumin (BSA) (1 mg/ml); CAT-protein from Neurospora crassa mitochondria (NCM) (1.6 mg/ml); CATprotein treated with 1% sodium dodecyl sulfate (1.5 mg/ml). Other conditions were as in Fig. 2.
[&-SICAT bound to the isolated CAT-protein can be displaced by dialysis against unlabeled CAT or ADP + BKA. Unlike the reaction with unlabeled CAT, the displacement of bound [35S]CAT by ADP + BKA may require the translocator to undergo a conformational transition (Fig. 5, reactions I and 2). The question arises whether the CAT-protein antibody, which reacts with the translocator in the c-state, can inhibit the conformational change induced by ADP + BKA. Such an inhibition would be reflected experimentally in the inhibition by a specific antibody of the (ADP + BKA)-promoted removal of CAT bound to the translocator. One prerequisite for this experiment would be that the antibody does not cover the CAT-binding site and therefore does not inhibit the removal of [asS]CAT by unlabeled CAT, as illustrated by scheme A in Fig. 5. The experimental results in Fig. 6 show that the rate of the removal of [asSICAT by unlabeled CAT was not inhibited by the CAT-protein antibody and indicate that the CAT-binding site is not made inaccessible by the antibody. However, the removal of [-5S]CAT with ADP + BKA was greatly inhibited by the antibody. This is indicative of an inhibition by the CAT-protein antibody of a conformational change that is required for binding of ADP + BKA, in accordance with scheme B in Fig. 5. Effect of antibodies on nucleotide transport In other experiments we attempted to determine whether the CAT-protein antibody inhibits mitochondrial nucleotide transport, as measured by the ADP,ATP exchange reaction (4, 5). A series of experiments was performed with beef heart mitochondria that were either untreated or treated in such a way as to render greater antibody accessibility to the bound translocator. There was in no case a significant inhibition by the antibody of the ADP,ATP exchange reaction in comparison with the control serum. DISCUSSION As previously deduced from studies with intact membranes, the mitochondrial ADP,ATP translocator can exist in two different states: the c-state (+CAT) and the m-state (+BKA). In coming to one of these states, the binding site of the translocator is oriented in one of two opposite positions with regard to the membrane surface (4, 6, 7). In the c-state, the binding site faces the cytosol side of the mitochondrion; in the m-state, the binding site faces the mitochondrial matrix. Concomitant with the orientation of the binding sites, a great change in affinity
~.
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan et al.
C-STATE
AB
ADP
BKA
22
21
C2= 1
'*-STATE
-
CAT
2283
A ANTIBODY COVERS
CAT BINDING
SITE
BKA , _ _ _
B ANTIBODY CLAMPS THE CONFORMATION
FIG. 5. Relation between the CAT-protein antibody and the possible conformational transitions associated with the displacement by BKA + ADP of protein-bound CAT. In case (A), the antibodies (AB) covering the CAT-binding site inhibit the removal of CAT in reaction (1), as measured by the exchange of [35S]CAT against unlabeled CAT. In case (B), antibodies clamp the translocator in a particular conformation without covering the binding site; although they do not affect the removal of CAT in reaction (1), the antibodies inhibit the removal of CAT that is associated with the transition from the c- to the m-state (reaction 2).
of the translocator for the high-affinity ligands CAT and BKA takes place (5, 9). The very high affinity of the c-state for CAT largely excludes the binding of BKA and, conversely, the high affinity of the m-state for BKA excludes the binding of CAT. Such behavior indicates the occurrence of drastic conformational changes that appear to be closely related to the overall transport process. The occurrence of specific conformations is also indicated by the finding that the sulfhydryl agent N-eth-
+AB
I .
-0 2(D ID
BKA ~0
+
ADP
1~~ ~ ~ ~ ~ ~
+CS
co 6C
+AB
Io,
4C
2C 2
4
6
8
24
hr
2
4
6
8
I
10 24
FIG. 6. Effect of CAT-protein antibody on the removal of ['5S]CAT from the [35S]CAT-protein by either CAT or BKA + ADP. The rate of dissociation of [35S]CAT was followed in an equilibrium dialyzer equipped with pairs of microdialysis cells. To each half-cell (I) was added [35S]CAT-protein and either excess antibody (AB) or control serum (CS). The opposing half-cell (II) contained the same buffer as in I [10 mM morpholinepropanesulfonic acid + 0.2 M NaCl (pH 7.6)]. At time 0, the displacing ligands were added in great excess (50 ,M CAT or 200,gM ADP + 50 AM BKA) to half-cell II. After dialysis was started, aliquots were withdrawn at the times indicated from both half-cells and assayed for released [u5SjCAT. Temperature, 18°. 0 half-cell I containing [35S]CAT protein; 0, half-cell II containing unlabeled CAT or ADP + BKA.
ylmaleimide reacts with the translocator only in the m-state (8, 12-14, and H. Aquila and M. Klingenberg, unpublished data). The present results with the isolated and purified translocator provide new evidence for the existence of specific conformational states of the ADP,ATP translocator. Evidence that the translocator has a particular conformation when isolated and purified as the CAT-protein complex came from the finding that this solubilized complex shows chromatographic behavior different from that of the unliganded protein (8, 9). The large differences in the antigenic properties of the isolated CATprotein and the BKA-protein strongly support the concept of a distinct conformational difference between the c- and mstates. Because the CAT-protein antibody interacts only weakly with the BKA-protein, and vice versa, it appears that nearly all of the antigenic determinants in one state are absent in the other state. This finding can best be explained by the conclusion that a specific conformational configuration gives the translocator its particular set of antigenic determinants. These determinants appear not to be concentrated at the binding centers, since the antibodies do not inhibit the translocator in situ and do not inhibit ligand exchange with the isolated CAT-protein. The determinants probably are located at the protein surface that becomes exposed after extraction of the protein from its phospholipid environment. Further evidence for the latter aspect and for the conformational specificity of the antibodies comes from the finding that the CAT-protein antibody, without directly interfering with the CAT-binding site, appears to immobilize the translocator in the specific conformation conferred by CAT. Thus, reactions that seem to require conformational changes, such as the removal of translocator-bound CAT by ADP + BKA, are inhibited by the antibodies. The Na+,K+-ATPase represents another example in which antibodies have been generated against a transport protein (14). In this connection, it is pertinent to note the findings of McCans et al. (15), which suggested that antibodies to canine renal ATPase were conformation-sensitive. Two different active antibody fractions were formed, but only one inhibited ATPase activity. This finding suggested the presence of a mixture of at least two conformations in the ATPase preparation. However, in that case the preparation was not fixed in a definite confor-
2284
Biochemistry: Buchanan et al.
mation prior to its use to generate antibodies. By contrast, in our experiments a purified transport protein fixed exclusively in one conformation was used to elicit the formation of antibodies that were specific for that conformation. This work was supported in part by a grant from the Sonderforschungsbereich 51 of the Deutsche Forschungsgemeinschaft. The support of a J. S. Guggenheim Fellowship to B.B.B. is gratefully acknowledged. 1. Klingenberg, M. (1976) "The ADP,ATP carrier in mitochondrial membranes," in The Enzymes of Biological Membranes (Plenum Publishing Corp., New York), Vol. 3, pp. 383-437. 2. Erdelt, H., Weidemann, M. J., Buchholz, M. & Klingenberg, M. (1972) "Some principle effects of bongkrekic acid on the binding of adenine nucleotides to mitochondrial membranes," Eur. J. Biochem. 30, 107-122. 3. Klingenberg, M. & Buchholz, M. (1973) "On the mechanism of bongkrekate effect on the mitochondrial adenine-nucleotide carrier as studied through the binding of ADP," Eur. J. Blochem. 38,346-358. 4. Klingenberg, M., Scherer, B., Stengel-Rutkowski, L., Buchholz, M. & Grebe, K. (1972) "Experimental demonstration of the reorienting (mobile) carrier mechanism exemplified by the mitochondrial adenine nucleotide translocator," in Mechanisms in Bioenergetics, eds. Azzone, G. F., Ernster, L., Papa, S., Quagliariello, E. & Siliprandi, N. (Academic Press, New York), pp.257-284. 5. Scherer, B. & Klingenberg, M. (1974) "Demonstration of the relationship between the adenine nucleotide carrier and the structural changes of mitochondria as induced by adenosine 5'diphosphate," Biochemistry 13, 161-170. 6. Klingenberg, M., Riccio, P., Aquila, H., Schmiedt, B., Grebe, K. & Topitsch, P. (1974) "Characterization of the ADP,ATP carrier in mitochondria," in Membrane Proteins in Transport and Phosphorylation, eds. Azzone, G. F., Klingenberg, M., Quagli-
Proc. Nati. Acad. Sci. USA 73 (1976)
7. 8. 9.
10.
11.
ariello, E. & Siliprandi, N. (North Holland Publishing Co., Amsterdam), pp. 229-243. Riccio, P., Aquila, H. & Klingenberg, M. (1975) "Solubilization of the carboxy-atractylate binding protein from mitochondria," FEBS Lett. 56, 129-132. Riccio, P., Aquila, H. & Klingenberg, M. (1975) "Purification of the carboxy-atractylate binding protein from mitochondria," FEBS Lett. 56,133-138. Klingenberg, M., Riccio, P., Aquila, H., Buchanan, B. B. & Grebe, K. (1976) "Mechanism of carrier transport and the ADP,ATP carrier," in The Structural Basis of Membrane Function, eds. Hatefi Y. & Djavadi-Ohaniance, L. (Academic Press, New York), pp. 293-311. Werner, S. (1974) "Isolation and characterization of a mitochondrially synthesized precursor protein of cytochrome oxidase," Eur. J. Biochem. 43,39-48. Masters, B. S. S., Baron, J., Taylor, W. E., Isaacson, E. L. & LoSpalluto, J. (1971) "Immunochemical studies on electron transport chains involving cytochromeP-450," J. Biol. Chem. 246,
4143-4150. 12. Leblanc, P., and Clauser, H. (1972) "ADP-dependent inhibition of sarcosomal adenine nucleotide translocase by N-ethylmaleimide," FEBS Lett. 23, 107-113. 13. Vignais, P. V. & Vignais, P. M. (1972) "Effect of SH reagents on attractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP," FEBS Lett. 26, 27-31. 14. Lauf, P. K. (1975) "Antigen-antibody reactions and cation transport in biomembranes: Immunophysiological aspects," Reviews on Biomembranes, Biochim. Biophys. Acta 415, 173-229. 15. McCans, J. L., Lane, L. K., Lindenmayer, G. E., Butler, V. P., Jr. & Schwartz, A. (1974) "Effects of an antibody to a highly purified Na+,K+-ATPase from canine renal medula: Separation of the H1oloenzyme Antibody into catalytic and cardiac glycoside receptor-specific components," Proc. Natl. Acad. Sci. USA 71, 2449-2452.
Antibody evidence for different conformational states of ADP,ATP translocator protein isolated from mitochondria (membranes/transport/protein conformation)
BOB B. BUCHANAN*, W. EIERMANN, P. RICCIOt, H. AQUILA, AND M. KLINGENBERGt Institute of Physiological Chemistry and Physical Biochemistry, University of Munich, Munich, Federal Republic of Germany
Communicated by Daniel I. Arnon, April 20,1976
stages in the transport of ADP or ATP because a transition between the m-state and the c-state occurs only during the translocation of one molecule of ADP or ATP. The knowledge of the existence of the translocator protein in two different orientation states was used to develop a procedure for the solubilization and purification of the translocator protein. The protein was first isolated after stabilization against denaturation by the formation of the CAT-protein complex (6-9), and was more recently isolated as the somewhat less stable BKA-protein complex (P. Riccio and M. Klingenberg, unpublished data). New evidence will be presented for two conformational states of the ADP,ATP translocator protein, based on the use of specific antibodies against the isolated ligand-protein complexes. The antibody against the CAT-protein complex characteristic of the c-state did not react with the translocator protein in the m-state. Conversely, the antibody against the BKA-protein characteristic of the m-state did not react with the protein in the c-state.
Consistent with the previously proposed reABSTRACT orientation mechanism for the ADPATP transl ator protein of mitochondria, evidence has now been obtained for the existence of two distinct conformational states of the isolated translocator protein. Previous studies indicated that when the mitochondrial translocator protein is in the c-state (i.e., when its binding site faces the cytosol side) the protein binds primarily the ligand carboxyatractylate (CAT), and when the translocator protein is in the m-state (i.e., when its binding site faces the mitochondrial matrix) the translocator protein binds primarily bongkrekate. Direct evidence for this formulation has now come from the application of antibodies to the isolated translocator
protein-ligand complex. Two antibodies were produced against the ADPATP translocator protein isolated from beef heart mitochondria. One antibody, which was produced against the protein isolated as the CAT-binding protein complex, was found to be highly specific for that complex and did not react with the protein in the conformation state conferred by the bongkrekate ligand. This antibody did not cover the CAT-bindingsite, as evidenced by the exchange of unlabeled CAT with [ SICAT bound to the translocator protein. However, the same antibody inhibited a transition of the protein from the c-state to the m-state, as evidenced by an inhibition of the displacement of [5S CAT by bofigkrekate (added jointly with ADP). It appears, therefore, that the antibody immobilized the translocator protein in the
METHODS Protein Purification. Methods for purification from beef heart mitochondria of the CAT-binding protein in Triton X-100 solutions were as described previously (7, 8). BKAbinding protein was solubilized from mitochondria loaded with ADP + BKA and purified by a similar procedure. The properties of the BKA-protein preparation will be reported elsewhere. Hydroxylapatite Fractions of Preloaded Mitochondria. Hydroxylapatite fractions containing the ADP,ATP translocator were obtained from "preloaded" mitochondria prepared by adding to 1 ml of thawed beef heart mitochondria (10 mg of protein) 40MgM of each of the following: (1) CAT; (2) ADP; (3) BKA; (4) ADP; and (5) ADP. Numbers (1) and (2) were incubated for 7 min without further additions; the following addition (to 40 MM) was made after 2 min to the remaining samples: (3) ADP; (4) BKA; and (5) CAT. Incubation of (3)-(5) was continued for an additional 5 min, and all samples were then centrifuged at 9000 X g for 15 min. The supernatant fraction was discarded and the translocator was solubilized by suspending the pellet in 1 ml of a solution (pH 7.2) containing 4% (vol/vol) Triton X-100, 20 mM 4-morpholinepropanesulfonic acid, and 0.5 M NaCl. After 1 hr, samples were centrifuged (1 hr at 143,000 X g), the pellet was discarded, and the supernatant fractions were applied to hydroxylapatite columns (0.8 X 12.5 cm) equilibrated beforehand with a solution (pH 7.2) containing 0.5% Triton X-100, 10 mM morpholinepropanesulfonic acid, and 100 mM NaCl. The translocator was then eluted with the same buffer solution. The translocator peak fractions (determined by sodium dodecyl sulfate gel electrophoresis) were combined and concentrated by ultrafiltration. Antibodies., For antibody production, rabbits received
c-state.
The second antibody produced against the (somewhat less pure) ADPATP translocator protein, isolated as the bongkrekate-binding protein complex, did not react with the CATbinding protein. Thus, the second antibody appeared to be specific for the translocator protein in the m-state. Neither antibody inhibited mitochondrial ADPATP transport. One feature of membranes central to their importance in living cells is their ability to catalyze a highly ordered transport of metabolites and ions by specific translocator proteins. In most eukaryotic cells, a key intracellular transport process is catalyzed by the ADP,ATP translocator protein (cf. ref. 1). Research in the last several years has considerably clarified the mode of action of the ADP,ATP translocator protein. It has been possible to demonstrate that the ADP,ATP binding site of the translocator protein is oriented either to the outer (cytosol) or to the inner (mitochondrial matrix) face of the membrane (2-5). Each of these states of protein orientation may be stabilized by the binding of a specific high-affinity ligand: carboxyatractylate (CAT) for the cytosol or c-state and bongkrekate (BKA) for the matrix or m-state. These two states appear to reflect different Abbreviations: CAT, carboxyatractylate; BKA, bongkrekate. * Permanent address: Department of Cell Physiology, University of California, Berkeley, Calif. 94720. t Present address: Istituto di Chimica Biologica, Universiti de Bari, Via Amendola 165A, 70126 Bari, Italy. t To whom reprint requests should be addressed: c/o Institut fur Physiologische Chemie und Physikalische Biochemie der Universitit Munchen, 8 Mfinchen 2, Goethestrasse 33, Federal Republic of Germany.
2280
Biochemistry:
Buchanan et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
.6
2281
CAT-PROT., BHM
'4
CATQ0
Ec 12 0 0
Tco
-.
1.0
'a 0.8
CAT+BSA
Antibody
C)
0
0
0o 0o
CAT-PROT RLM CAT-PROT NCM
CAT+DPG.BSA
o 0.4 Anti body (0.5% Triton)
0.2 --.A6...
o
20
30
40
50
60
70
80
90
M1 rGIbt~n FIG. 1. Precipitation of beef heart mitochondrial CAT-protein by its antibody. Antibody or control gamma globulin (69 mg/ml) was added stepwise to a 1 ml cuvette (1 cm light path) containing 0.6 ml of 56 mM sodium barbital buffer supplemented with 56 mM sodium acetate (pH 8.4) and 98;tg of CAT-protein that was dissolved in 0.2 ml of a solution containing 20 mM morpholinepropanesulfonic acid buffer, 50 mM NaCl, and 0.5% Triton X-100 (pH 7.2). Additional Triton X-100 (0.5%) was added to the indicated sample. Optical density was measured at 600 nm. Time between gamma globulin additions was 45 min. Length of experiment was 14 hr.
through their rear footpads the CAT-translocator complex in solution (pH 7.2) containing 5 mM morpholinepropanesulfonic acid, 50 mM NaCl, and the small amount of Triton X-100 that remained after extraction of the original 5% Triton solution three times with diethyl ether. The BKA-translocator complex was injected into rabbits in a solution containing 5% Triton X-100. Prior to injection, an emulsion of incomplete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) was added to the protein solution in a volume ratio of 3:1 (10). The initial injection of 2 mg of protein was followed after 3 days by a booster injection of 1 mg of protein and by four subsequent booster injections, each of 1 mg of protein, administered at weekly intervals. Antibody was detected in the serum by the Ouchterlony double-diffusion technique after 1 month. The serum was then collected biweekly over a 2-week period, after which the rabbits were killed and bled for collection of total serum. The gamma globulin fraction was isolated from the pooled sera of each rabbit by ammonium sulfate fractionation and DEAE-cellulose chromatography (11). Control serum collected from the same animals prior to injection was fractionated in the same manner. Other Methods. Methods for sodium dodecyl sulfate gel electrophoresis, preparation of [35S]CAT, and for estimation of protein were as described previously (3). a
RESULTS Prior to its solubilization and purification for the experiments reported below, the ADP,ATP translocator was fixed in either of its two orientations in the membrane by binding to it one of the high-affinity ligands, CAT or BKA (2, 3). Because of their very high affinity for the translocator (Kd < 10-10 M), these ligands stabilize the translocator in a specific orientation to a much greater extent than does ADP or ATP. After binding CAT, the binding site of the translocator faces the outer (cytosol) surface of the membrane ("c-state"); after binding BKA, the binding site faces the inner (matrix) surface of the membrane ("m-state") (6). The orientations conferred by these ligands appear to be retained during the procedures used for the solubilization and purification of the translocator.
FIG. 2. Specificity of antibody against the beef heart mitochondrial CAT-protein in Ouchterlony double diffusion. The wells and precipitin band are shown in a high-contrast negative of the Ouchterlony plate. Center well, 5 1d of CAT-protein antibody (AB) (37 mg/ml). Outer wells, 5 ,d of one of the following: CAT-protein (2 mg/ml) from beef heart mitochondria (BHM); partly purified CATprotein (2 mg/ml) from rat liver mitochondria (RLM); CAT-protein (2 mg/ml) from Neurospora crassa mitochondria (NCM); or the equivalent amount of free CAT (40MuM), or CAT (40 MM) + bovine serum albumin (BSA) (2 mg/ml), or CAT (40 MM) + cardiolipin (DPG) (0.03 mg/ml) + BSA (2 mg/ml). The only Triton X-100 present was that added to the outer wells with the 5 ,l samples of the CATprotein or to the other agents tested for antigenic activity. Each of these solutions contained 0.5% Triton X-100.
Antibody against the CAT-translocator complex Fig. 1 demonstrates the presence of an antibody against the purified ADP,ATP translocator that was fixed in the c-state by the high-affinity ligand CAT (6). The presence of the antibody was demonstrated by its ability to precipitate specifically the CAT-translocator complex (henceforth called the CAT-protein). Precipitation was sensitive to the detergent Triton X-100 and was nearly completely inhibited by the 0.5% concentration routinely used to maintain the CAT-protein in solution. The inhibition by Triton X-100 was found in other experiments to be due not to interference of binding of the antibody to the translocator but to inhibition of precipitate formation after binding had occurred. In these experiments, the free antibody that remained after incubation with the CAT-protein dissolved in different concentrations of Triton X-100 was estimated by its capacity to precipitate a second aliquot of the CAT-protein in Ouchterlony double diffusion (cf. Fig. 2). To determine its specificity, the antibody against the CATprotein from beef heart mitochondria was tested for its ability to precipitate other proteins. As shown in Fig. 2, the antibody reacted only with its specific antigen when tested by the Ouchterlony double-diffusion technique. (Positive reaction is indicated by the presence of a precipitin line.) There was no reaction of antibody with fresh preparations of the pure CAT-protein from Neurospora crassa mitochondria or with the partially purified counterpart from rat liver mitochondria. Also, CAT, alone or in the presence of bovine serum albumin or cardiolipin, gave no precipitin reaction. The inability of antibodies against the CAT-protein from beef heart mitochondria to react with the CAT-protein from other sources or with CAT bound to bovine serum albumin indicates that the translocator protein serves as the antigenic determinant in antibody formation, independently of the bound CAT. Further evidence that CAT itself does not elicit antibody formation (i.e., function as a hapten group) was provided by the findings that the CAT-protein antibody did not bind [a5S]CAT in equilibrium dialysis and that added CAT had no effect on precipitation of the CAT-protein in Ouchterlony double-diffusion experiments. The specificity of the antibody for a particular conformation of the translocator was shown in a comparison of its reactivity
2282
Biochemistry: Buchanan et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
UNLOADED
BKA-PROT BHM
0
ADP-BKA
0
CAT-LOADED
v
ADP
BKA-ADP
FIG. 3. Reaction of CAT-protein antibody with the translocator from mitochondria preloaded with different ligands. Center well, 5 !d of CAT-protein antibody (69 mg/ml). Outer wells, 5 ,d of one of the following: translocator preparations (hydroxylapatite column passthrough fractions) that were preloaded as indicated with CAT, ADP, BKA + ADP, ADP + BKA, or ADP + CAT. The column passthrough fractions contained approximately equal amounts of the translocator as determined by sodium dodecyl sulfate gel electrophoresis. Other conditions were as in Fig. 2.
with preparations obtained from beef heart mitochondria preloaded with different ligands. Fig. 3 shows that, of several different ligands (including BKA), the antibody precipitated only the translocator preparation derived from mitochondria preloaded with CAT. Other experiments revealed that the antibody did not react with the derivatives of the CAT-protein obtained after treatment with sodium dodecyl sulfate or ATP (unpublished data). We conclude, therefore, that the antibodies are specific not only for the particular translocator native to beef heart mitochondria but also for a specific conformation of that translocator that is conferred by CAT. The conferral of a specific conformation to the ADP,ATP translocator on binding CAT is also substantiated by other properties of the isolated protein: in contrast to the nonliganded protein, the CAT-protein in Triton X-100 solution is (i) not adsorbed strongly on hydroxylapatite, and (ii) maintained in a state largely protected against denaturation and subsequent degradation by proteinases (P. Riccio and M. Klingenberg, unpublished data). The BKA-translocator complex appears to be considerably less stabilized in its conformation. Antibody against the BKA-translocator complex Because the translocator would be expected, on the basis of earlier findings, to become fixed in a different, well-defined conformation on binding BKA (m-state) (5, 9), we attempted to obtain an antibody against the purified BKA-translocator complex (henceforth called the BKA-protein). The isolated BKA-protein complex appears to contain the same polypeptide chain as the CAT protein (P. Riccio and M. Klingenberg, unpublished results) and therefore, in agreement with the postulates of the reorientation mechanism, can be assumed to represent another conformation of the same protein. Fig. 4 shows not only that antibodies were produced against the BKA-protein, but also that they were quite specific: a precipitin reaction was observed only with the antigen and not with the CAT-protein or its sodium dodecyl sulfate derivative. In no case was there a reaction between the BKA-protein or the CATprotein and the control gamma globulin fraction obtained with rabbits prior to immunization. Effect of antibodies on change in translocator conformation As sensitive indicators of conformation, the antibodies against the translocator might be expected to influence the conformational changes that accompany transition of the protein between the c- and m-states. It is known from earlier work that
Sodim dodecyl s heated CAT-PROT. CAT- PROT NCM
0
0
0
00 0
CAT- PROT BHM
BKA+BSA
BKA+DPG+BSA FIG. 4. Specificity of antibody against the beef heart mitochondrial BKA-protein. Center well, 5 ,g of BKA-protein antibody (40 mg/ml). Outer wells, 5 ,g of one of the following: BKA-protein from beef heart mitochondria (BHM) (1.2 mg/ml); CAT-protein from beef heart mitochondria (1.5 mg/ml); BKA (50 AsM) + cardiolipin (DPG) (0.03 mg/ml) + bovine serum albumin (BSA) (1 mg/ml); CAT-protein from Neurospora crassa mitochondria (NCM) (1.6 mg/ml); CATprotein treated with 1% sodium dodecyl sulfate (1.5 mg/ml). Other conditions were as in Fig. 2.
[&-SICAT bound to the isolated CAT-protein can be displaced by dialysis against unlabeled CAT or ADP + BKA. Unlike the reaction with unlabeled CAT, the displacement of bound [35S]CAT by ADP + BKA may require the translocator to undergo a conformational transition (Fig. 5, reactions I and 2). The question arises whether the CAT-protein antibody, which reacts with the translocator in the c-state, can inhibit the conformational change induced by ADP + BKA. Such an inhibition would be reflected experimentally in the inhibition by a specific antibody of the (ADP + BKA)-promoted removal of CAT bound to the translocator. One prerequisite for this experiment would be that the antibody does not cover the CAT-binding site and therefore does not inhibit the removal of [asS]CAT by unlabeled CAT, as illustrated by scheme A in Fig. 5. The experimental results in Fig. 6 show that the rate of the removal of [asSICAT by unlabeled CAT was not inhibited by the CAT-protein antibody and indicate that the CAT-binding site is not made inaccessible by the antibody. However, the removal of [-5S]CAT with ADP + BKA was greatly inhibited by the antibody. This is indicative of an inhibition by the CAT-protein antibody of a conformational change that is required for binding of ADP + BKA, in accordance with scheme B in Fig. 5. Effect of antibodies on nucleotide transport In other experiments we attempted to determine whether the CAT-protein antibody inhibits mitochondrial nucleotide transport, as measured by the ADP,ATP exchange reaction (4, 5). A series of experiments was performed with beef heart mitochondria that were either untreated or treated in such a way as to render greater antibody accessibility to the bound translocator. There was in no case a significant inhibition by the antibody of the ADP,ATP exchange reaction in comparison with the control serum. DISCUSSION As previously deduced from studies with intact membranes, the mitochondrial ADP,ATP translocator can exist in two different states: the c-state (+CAT) and the m-state (+BKA). In coming to one of these states, the binding site of the translocator is oriented in one of two opposite positions with regard to the membrane surface (4, 6, 7). In the c-state, the binding site faces the cytosol side of the mitochondrion; in the m-state, the binding site faces the mitochondrial matrix. Concomitant with the orientation of the binding sites, a great change in affinity
~.
Proc. Natl. Acad. Sci. USA 73 (1976)
Biochemistry: Buchanan et al.
C-STATE
AB
ADP
BKA
22
21
C2= 1
'*-STATE
-
CAT
2283
A ANTIBODY COVERS
CAT BINDING
SITE
BKA , _ _ _
B ANTIBODY CLAMPS THE CONFORMATION
FIG. 5. Relation between the CAT-protein antibody and the possible conformational transitions associated with the displacement by BKA + ADP of protein-bound CAT. In case (A), the antibodies (AB) covering the CAT-binding site inhibit the removal of CAT in reaction (1), as measured by the exchange of [35S]CAT against unlabeled CAT. In case (B), antibodies clamp the translocator in a particular conformation without covering the binding site; although they do not affect the removal of CAT in reaction (1), the antibodies inhibit the removal of CAT that is associated with the transition from the c- to the m-state (reaction 2).
of the translocator for the high-affinity ligands CAT and BKA takes place (5, 9). The very high affinity of the c-state for CAT largely excludes the binding of BKA and, conversely, the high affinity of the m-state for BKA excludes the binding of CAT. Such behavior indicates the occurrence of drastic conformational changes that appear to be closely related to the overall transport process. The occurrence of specific conformations is also indicated by the finding that the sulfhydryl agent N-eth-
+AB
I .
-0 2(D ID
BKA ~0
+
ADP
1~~ ~ ~ ~ ~ ~
+CS
co 6C
+AB
Io,
4C
2C 2
4
6
8
24
hr
2
4
6
8
I
10 24
FIG. 6. Effect of CAT-protein antibody on the removal of ['5S]CAT from the [35S]CAT-protein by either CAT or BKA + ADP. The rate of dissociation of [35S]CAT was followed in an equilibrium dialyzer equipped with pairs of microdialysis cells. To each half-cell (I) was added [35S]CAT-protein and either excess antibody (AB) or control serum (CS). The opposing half-cell (II) contained the same buffer as in I [10 mM morpholinepropanesulfonic acid + 0.2 M NaCl (pH 7.6)]. At time 0, the displacing ligands were added in great excess (50 ,M CAT or 200,gM ADP + 50 AM BKA) to half-cell II. After dialysis was started, aliquots were withdrawn at the times indicated from both half-cells and assayed for released [u5SjCAT. Temperature, 18°. 0 half-cell I containing [35S]CAT protein; 0, half-cell II containing unlabeled CAT or ADP + BKA.
ylmaleimide reacts with the translocator only in the m-state (8, 12-14, and H. Aquila and M. Klingenberg, unpublished data). The present results with the isolated and purified translocator provide new evidence for the existence of specific conformational states of the ADP,ATP translocator. Evidence that the translocator has a particular conformation when isolated and purified as the CAT-protein complex came from the finding that this solubilized complex shows chromatographic behavior different from that of the unliganded protein (8, 9). The large differences in the antigenic properties of the isolated CATprotein and the BKA-protein strongly support the concept of a distinct conformational difference between the c- and mstates. Because the CAT-protein antibody interacts only weakly with the BKA-protein, and vice versa, it appears that nearly all of the antigenic determinants in one state are absent in the other state. This finding can best be explained by the conclusion that a specific conformational configuration gives the translocator its particular set of antigenic determinants. These determinants appear not to be concentrated at the binding centers, since the antibodies do not inhibit the translocator in situ and do not inhibit ligand exchange with the isolated CAT-protein. The determinants probably are located at the protein surface that becomes exposed after extraction of the protein from its phospholipid environment. Further evidence for the latter aspect and for the conformational specificity of the antibodies comes from the finding that the CAT-protein antibody, without directly interfering with the CAT-binding site, appears to immobilize the translocator in the specific conformation conferred by CAT. Thus, reactions that seem to require conformational changes, such as the removal of translocator-bound CAT by ADP + BKA, are inhibited by the antibodies. The Na+,K+-ATPase represents another example in which antibodies have been generated against a transport protein (14). In this connection, it is pertinent to note the findings of McCans et al. (15), which suggested that antibodies to canine renal ATPase were conformation-sensitive. Two different active antibody fractions were formed, but only one inhibited ATPase activity. This finding suggested the presence of a mixture of at least two conformations in the ATPase preparation. However, in that case the preparation was not fixed in a definite confor-
2284
Biochemistry: Buchanan et al.
mation prior to its use to generate antibodies. By contrast, in our experiments a purified transport protein fixed exclusively in one conformation was used to elicit the formation of antibodies that were specific for that conformation. This work was supported in part by a grant from the Sonderforschungsbereich 51 of the Deutsche Forschungsgemeinschaft. The support of a J. S. Guggenheim Fellowship to B.B.B. is gratefully acknowledged. 1. Klingenberg, M. (1976) "The ADP,ATP carrier in mitochondrial membranes," in The Enzymes of Biological Membranes (Plenum Publishing Corp., New York), Vol. 3, pp. 383-437. 2. Erdelt, H., Weidemann, M. J., Buchholz, M. & Klingenberg, M. (1972) "Some principle effects of bongkrekic acid on the binding of adenine nucleotides to mitochondrial membranes," Eur. J. Biochem. 30, 107-122. 3. Klingenberg, M. & Buchholz, M. (1973) "On the mechanism of bongkrekate effect on the mitochondrial adenine-nucleotide carrier as studied through the binding of ADP," Eur. J. Blochem. 38,346-358. 4. Klingenberg, M., Scherer, B., Stengel-Rutkowski, L., Buchholz, M. & Grebe, K. (1972) "Experimental demonstration of the reorienting (mobile) carrier mechanism exemplified by the mitochondrial adenine nucleotide translocator," in Mechanisms in Bioenergetics, eds. Azzone, G. F., Ernster, L., Papa, S., Quagliariello, E. & Siliprandi, N. (Academic Press, New York), pp.257-284. 5. Scherer, B. & Klingenberg, M. (1974) "Demonstration of the relationship between the adenine nucleotide carrier and the structural changes of mitochondria as induced by adenosine 5'diphosphate," Biochemistry 13, 161-170. 6. Klingenberg, M., Riccio, P., Aquila, H., Schmiedt, B., Grebe, K. & Topitsch, P. (1974) "Characterization of the ADP,ATP carrier in mitochondria," in Membrane Proteins in Transport and Phosphorylation, eds. Azzone, G. F., Klingenberg, M., Quagli-
Proc. Nati. Acad. Sci. USA 73 (1976)
7. 8. 9.
10.
11.
ariello, E. & Siliprandi, N. (North Holland Publishing Co., Amsterdam), pp. 229-243. Riccio, P., Aquila, H. & Klingenberg, M. (1975) "Solubilization of the carboxy-atractylate binding protein from mitochondria," FEBS Lett. 56, 129-132. Riccio, P., Aquila, H. & Klingenberg, M. (1975) "Purification of the carboxy-atractylate binding protein from mitochondria," FEBS Lett. 56,133-138. Klingenberg, M., Riccio, P., Aquila, H., Buchanan, B. B. & Grebe, K. (1976) "Mechanism of carrier transport and the ADP,ATP carrier," in The Structural Basis of Membrane Function, eds. Hatefi Y. & Djavadi-Ohaniance, L. (Academic Press, New York), pp. 293-311. Werner, S. (1974) "Isolation and characterization of a mitochondrially synthesized precursor protein of cytochrome oxidase," Eur. J. Biochem. 43,39-48. Masters, B. S. S., Baron, J., Taylor, W. E., Isaacson, E. L. & LoSpalluto, J. (1971) "Immunochemical studies on electron transport chains involving cytochromeP-450," J. Biol. Chem. 246,
4143-4150. 12. Leblanc, P., and Clauser, H. (1972) "ADP-dependent inhibition of sarcosomal adenine nucleotide translocase by N-ethylmaleimide," FEBS Lett. 23, 107-113. 13. Vignais, P. V. & Vignais, P. M. (1972) "Effect of SH reagents on attractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP," FEBS Lett. 26, 27-31. 14. Lauf, P. K. (1975) "Antigen-antibody reactions and cation transport in biomembranes: Immunophysiological aspects," Reviews on Biomembranes, Biochim. Biophys. Acta 415, 173-229. 15. McCans, J. L., Lane, L. K., Lindenmayer, G. E., Butler, V. P., Jr. & Schwartz, A. (1974) "Effects of an antibody to a highly purified Na+,K+-ATPase from canine renal medula: Separation of the H1oloenzyme Antibody into catalytic and cardiac glycoside receptor-specific components," Proc. Natl. Acad. Sci. USA 71, 2449-2452.
