JOURNAL OF BACTERIOLOGY, Oct. 1975, p. 470-475 Copyright © 1975 American Society for Microbiology
Vol. 124, No. 1 Printed in U.S.A.
Localization of D-Lactate Dehydrogenase in Membrane Vesicles Prepared by Using a French Press or Ethylenediaminetetraacetate-Lysozyme from Escherichia coli MASAMITSU FUTAI* AND YASUHITO TANAKA Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan Received for publication 29 May 1975
The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibcody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane. Membrane vesicles prepared from Escherichia coli by using a French press (3, 9) and by treatment with ethylenediaminetetraacetate (EDTA)-lysozyme as described by Kaback (10) have been used for studies on oxidative phosphorylation (9) and active transport (11), respectively. We have suggested that membrane vesicles prepared by using a French press are inverted, as adenosine triphosphatase (ATPase), its binding sites, and ferricyanide reductase were found outside the membranes (3). Ferricyanide was formerly used as an impermeant electron acceptor from dehydrogenases (22). However, it is not a good electron acceptor for purified D-lactate dehydrogenase (4). This suggests that ferricyanide accepts electrons from a component(s) in the respiratory chain other than the dehydrogenase in the n-lactate oxidase system. Thus it seemed interesting to study the location of dehydrogenase itself in French press vesicles (membrane vesicles prepared by using a French press as described [31) by using immunochemical procedures.
Based on the above criteria we have also suggested that the orientation of half the membrane vesicles prepared by treatment with EDTA-lysozyme are changed from those of intact cells or spheroplasts (3). Evidence from other laboratories also suggests that EDTAlysozyme vesicles are a heterogeneous population (6, 7, 21). However, Kaback and his associates claimed that these vesicles are a homogenous right-side out population (10, 18, 19). Rosen and McClees demonstrated that EDTA-lysozyme vesicles did not transport calcium, whereas inverted French press vesicles do (16). Therefore, since EDTA-lysozyme vesicles are widely used for studies on transport, it is important to study the location of enzymes to determine the orientation of their membrane definitely. Topological information is also important for understanding the transport mechanism, as already pointed out by Harold (8). D-Lactate dehydrogenase (D-LDH), which is a membrane-bound primary dehydrogenase in
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ORIENTATION OF MEMBRANE VESICLES FROM E. COLI
471
the respiratory chain of E. coli, was solubilized Spheroplasts or membranes were treated with 1% and purified to homogeneity (2, 12). Recently toluene as described previously (3). The activity of was measured as phenazine methosulfate-couShort et al. reported that D-LDH in EDTA- D-LDHreduction of MTT (D-lactate:MTT reduction) lysozyme vesicles was not accessible by its pled (2). Ferricyanide reduction (3), proline transport (4), antibody, concluding that these vesicles are a oxygen uptake (4), and protein (13) were assayed as homogeneous right-side-out population (17, 19). described previously. Thus it is of interest to study the location of Materials. Pure i)-LDH was obtained as described D-LDH in EDTA-lysozyme vesicles prepared by previously (2). The chemicals used were as described us, as we have repeatedly observed their previously (2-4) or were the highest grade commercially available. heterogeneity (3, 22). In this paper the location of D-LDH in memRESULTS brane vesicles prepared from E. coli by using a anti-D-LDH. The antibody Properties of French press and by treatment with EDTAlysozyme was studied by using antibody against prepared against D-LDH was specific for memthe purified enzyme. Our results on EDTA- brane-bound D-LDH purified from E. coli ML lysozyme vesicles essentially agreed with Short 308-225. As shown in Fig. 1, on immunodiffuet al. (19), as a majority (85%) of the activity of sion a precipitin line was observed between D-lactate-dependent oxygen uptake and 3-(4,5dimethyl-2)2,5 diphenyl tetrazolium bromide (MTT) reduction was not inhibited by the antibody and none of the D-lactate-dependent proline uptake was inhibited. However, unlike them a small portion of activity (15%) of D-lactate-dependent oxygen uptake and D-lactate:MTT reduction was inhibited. These results are discussed in relation to the location of 6 ferricyanide reductase and to our previous findings (3, 22). MATERIALS AND METHODS Bacteria and growth conditions. E. coli ML308225 (i- z- y+ a+) was grown aerobically in synthetic medium (20) with 0.5% D,L-lactate as a carbon source, unless otherwise specified. 5 Preparation of membrane vesicles. Membrane
~2
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4s
vesicles were prepared from EDTA-lysozyme spheroplasts as described by Kaback (10) using an 18-gauge needle for homogenization (19). Vesicles were also obtained by disruption of cells in a French press (pressure set, about 200 kg/cm2) (3). Both types of vesicles were used on the day of their preparation.
Preparation of antibody against I-LDH. Albino rabbits (about 1.5 kg) were immunized with 0.1 mg of purified D-LDH in complete Freund adjuvant (Difco Laboratories). Twenty days later they were injected with 0.05 mg of antigen in the same adjuvant. Thirty and 40 days after the second injection, they were injected with 0.05 mg of antigen in incomplete Freund adjuvant. For the injections, the antigen in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) containing 0.5% cholate was mixed with an equal volume of adjuvant. The mixture was injected subcutaneously. Animals were bled before immunization and 5 to 6 days after each booster injection. Antisera were examined by immunodiffusion (15). A precipitin line was usually detected after 4 or 5 booster injections, and gamma globulin was purified from the sera by ammonium sulfate precipitation and diethylaminoethyl-cellulose chromatography (13). Other procedures. Spheroplasts were prepared by using EDTA-lysozyme as described previously (3).
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-
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FIG. 1. Immunodiffusion pattern of antiserum to D-LDH. Antiserum diluted fourfold with saline was placed in the center well and different proteins and detergents were placed in the peripheral wells. (1) Purified D-LDH (0.11 mg of protein/ml) in 0.05 M tris(hydroxymethyl)aminomethane (pH 7.5) containing 1.0%o Triton X-100. (2) Purified D-LDH (0.11 mg/ml) in the above buffer without Trition X-100. (3) Deoxycholate extract of membranes from ML 308-225 (4.5 mg/ml). (4) 1.0%o Triton X-100. (5) 1.0% cholate. (6) Supernatant solution obtained after lysis of spheroplasts in 0.07 M tris(hydroxymethyl)aminomethane buffer, pH 8.0 (26 mg/ml). About 5 Al of each sample was placed in the wells. Serum from preimmune rabbits did not form a precipitin line with purified D-LDH or deoxycholate extract (data not shown). After diffusion the plate was dried and stained with amido black.
472
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FUTAI AND TANAKA
anti-D-LDH and purified D-LDH or an extract of the membane with deoxycholate. No reaction was observed with the supernatant fraction obtained after lysis of the spheroplasts. Only one precipitin line was observed between anti-DLDH and membrane in 2% Triton X-100 in agarose containing the same concentration of Triton X-100 (data not shown). This suggests that the preparation of anti-D-LDH did not contain antibodies to other membrane proteins. Addition of anti-D-LDH to purified D-LDH caused time-dependent inhibition of activity; D-LDH was inhibited 90% on incubation with 200 ,ug of anti-D-LDH for 16 h (Fig. 2A). On the other hand, the r-globulin fraction obtained from rabbit before immunization had no effect on the activity and did not form a precipitin line with D-LDH in the immunodiffusion test. Excess anti-D-LDH (0.8 or 1.7 mg) had no effect on 1 U of L-LDH purified from E. coli ML 308-225 (dld-) (H. Kimura and M. Futai, manuscript in preparation), suggesting the specificity of the antibody. Effect of anti-D-LDH on the i-LDH activity in spheroplasts. As shown in Table 1, D-LDH activity in spheroplasts was not sensitive to anti-D-LDH when the spheroplasts were protected by sucrose. In this experiment, spheroplasts were preincubated for 1 h with anti-DLDH, as keeping them intact for more than 2 h was difficult even in the presence of sucrose. However, D-LDH activity became sensitive to the antibody when the spheroplasts were treated with toluene and preincubated with anti-D-LDH as above. On the other hand anti-DLDH had no effect on the activities of other dehydrogenases assayed in toluenized spheroplasts (see Table 1). These results suggest that D-LDH is localized on the inside of the cytoplasmic membrane and reduces the permeable dye used in the assay mixture. The activity of D-LDH in spheroplasts which had been treated with toluene was inhibited by the antibody less than that of purified enzyme or enzyme in membrane vesicles prepared by using a French press. This may be because the time of incubation was too short or the permeability of toluene-treated spheroplasts was too low. Effect of anti-z-LDH on membrane vesicles prepared by using a French press. Membranes were preincubated for 16 h in the following experiments, although substantial inhibition was observed even on incubation for 30 min. All the activities tested below were retained during preincubation in the absence of
antibody. Addition of anti-D-LDH to membrane vesicles prepared by using a French press caused
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FIG. 2. Effect of anti-D-LDH on purified D-LDH and membrane vesicles prepared by using a French press or treatment with EDTA-lysozyme. (A) Purified D-LDH (1.0 U) was incubated with different amounts of anti-D-LDH for 30 min (0) and 16 h (0) at 0 C in 0.2 ml of 0.05 M potassium phosphate buffer (pH 6.6) containing 0.23% Triton X-100, and D-lactate:MTT reduction was assayed. Activities are expressed as percentage of that of the control. (B) Membrane vesicles prepared by using a French press were incubated for 16 h at 0 C with different concentrations of anti-D-LDH in 0.22 ml of 0.05 Mpotassium phosphate buffer, pH 6.6. After incubation D-lactate:MTT reduction (A) and D-lactate-dependent oxygen uptake (0) were assayed and expressed as percentage of those of the controls (without antibody). The control rates of D-lactate:MTT reduction and D-lactate-dependent oxygen uptake were 0.28 Mmol and 108 ng of atom 0, respectively, per min per mg of protein. (As controls L-lactate, succinate, or glycerol3-phosphate dehydrogenase in French press vesicles were assayed under the same conditions. None of those activities was sensitive to anti-D-LDH up to 1.0 mg of protein/U of each enzyme. (C) Membrane vesicles prepared by treatment with EDTA-lysozyme as described by Kaback (10) and Short et al. (19) were incubated with anti-D-LDH in 0.05 M potassium phosphate buffer, pH 6.6, for 16 h at 0 C. After incubation D-lactate:MTT reductase (A), D-lactatedependent oxygen uptake (0), and proline uptake (U) were assayed. Activities are expressed as percentage of those of the control (without antibody). The control rates per minute per milligram of protein were: D-lac-
tate:MTT reductase, 0.45 ;tmol; D-lactate-dependent
oxygen uptake, 449 ng of atom 0; D-lactate-dependent proline uptake, 1.0 nmol.
almost complete inhibition of D-lactate-dependent reduction of MTT and oxygen uptake (Fig. 2B). D-Lactate-dependent reduction of ferricyanide was also inhibited to the same extent (data not shown). These results are in good agreement with our previous observations (3). Activities of other dehydrogenases in French press vesicles were not sensitive to anti-D-LDH, suggesting the inhibition observed above is specific (legend of Fig. 2B). Effects of anti-D-LDH on membrane vesi-
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VOL. 124, 1975
TABLE 1. Effectofanti-D-LDHonD-LDHactivityin spheroplastsa D-LDH activity Anti-D-LDH
(4g) 0 50 100 200
(jAmol of MTT reduced/mg per min) Untreated
Treated with toluene
0.087 0.082 0.075 0.082
0.085 0.050 0.054 0.040
a Spheroplasts were obtained as described previously (3) except that 1 g (wet weight) of cells was suspended in 20 ml of a 20% sucrose solution. Spheroplasts (1.1 mg of protein) in 0.2 ml of 10% sucrose containing 0.01 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) were preincubated for 1 h at 20 C with different concentrations of anti-D-LDH. D-Lactate:MTT reduction was assayed in 10% sucrose using 10-MA aliquots of each preincubation mixture. Since spheroplast suspensions containing EDTA and lysozyme were directly used in the above assay, the concentration of cell protein was corrected for the amount of lysozyme added. One unit of L-lactate, succinate, or glycerol-3-phosphate dehydrogenase in spheroplasts treated with toluene was not inhibited by anti-D-LDH (up to 1.0 mg).
cles prepared by treatment with EDTA-lysozyme. As shown in Fig. 2C, D-lactate-dependent oxidation and D-lactate:MTT reduction in membrane vesicles prepared by treatment with EDTA-lysozyme were only slightly inhibited (about 15%) by the antibody. Furthermore, no inhibition of proline transport stimulated by D-lactate was observed. Short et al. (19) reported that neither of these activities were inhibited. The slight inhibition of D-lactate oxidation and D-lactate:MTT reduction we observed may be due to a difference in our preparation from theirs. Even with a large excess of antibody, less than 15% of the D-lactate oxidase activity and D-lactate:MTT reduction in this particular preparation were inhibited (Fig. 2C), although these activities were completely inhibited by antibody after sonic treatment of these vesicles (data not shown). We have repeated this titration with four different preparations of EDTA-lysozyme vesicles. The results suggested that the slight inhibitions of both activities observed by us are significant: (i) 400 and 800 ug of anti-D-LDH inhibited D-lactate-dependent oxygen uptake 15 i 5.3 and 14 :i 4.0%, respectively; (ii) 400 and 800 ug of anti-D-LDH inhibited D-lactate:MTT reduction 15 i 3.0 and 12 i 5.4%, respectively. D-Lactate-dependent uptake of proline by these different preparations was not sensitive to antiD-LDH. The interpretation of these results will be discussed below.
473
Ferricyanide reductase of membrane vesicles prepared by treatment with EDTAlysozyme. Ferricyanide reductase activity was not measurable in spheroplasts, but nearly half the total was detected in membrane vesicles prepared by EDTA-lysozyme using a PotterElvjem homogenizer (3, 22). Ferricyanide was suggested to be nonpermeable to the membranes of E. coli, since it inhibited transport into membrane vesicles stimulated by external reduced nicotinamide adenine dinucleotide, but not that stimulatd by internally generated reduced nicotinamide adenine dinucleotide (5). In this study membranes were dispersed with hypodermic needles instead of a homogenizer. This modification was made recently by Short et al. (19), who showed that it minimized the movement of ATPase from inside to outside the membrane during isolation of vesicles. Therefore, it seemed of interest to assay ferricyanide reduction by these membranes after preincubation in the presence or absence of toluene, which destroyed the permeability barrier for ferricyanide. As shown in Table 2, the activity of ferricyanide reductase was detected using glycerol3-phosphate, succinate, or D-lactate as substrate, and it increased about two- to threefold after the membranes had been treated with toluene. Toluene had no effect on the activity in membrane vesicles prepared by using a French TABLE 2. Ferricyanide reductase activity of membrane vesicles prepared by treatment with EDTA-lysozyme using a hypodermic needle for homogenizationa Ferricyanide reduction Substrate
(ismol reduced/mg per min) Untreated
Treated with toue toluene
Glycerol-3-
phosphate Succinate D-Lactate
0.049 0.017 0.026
0.140
0.034 0.056
aMembrane vesicles were prepared from E. coli ML 308-225 grown on 0.5% glycerol by the procedure of Kaback (10). A hypodermic needle was used to disperse membranes and spheroplasts throughout the preparation (19). Reduction of ferricyanide was followed spectrophotometrically at 420 nm in the presence of each substrate (20 mM). The increase of each activity of membrane vesicles prepared by using a French press could not be observed after toluene treatment. These results were confirmed by colorimetric assay using bathophenanthroline sulfonate (L. A. Heppel, personal communication). Other procedures were described in the text of a previous paper
(3).
474
FUTAI AND TANAKA
press, as shown previously (3). These results suggest that about 30 to 50% of ferricyanide sites are accessible, confirming our results on membranes prepared using a Potter-Elvjem homogenizer (3; L. A. Heppel confirmed this observation independently using membrane vesicles prepared from E. coli using a hypodermic needle for homogenization). It should be noted that this activity is not necessarily due to dehydrogenase, as discussed above, but it is provisionally assumed that it is due to a ferricyanide site(s), which is inaccessible from the outside of the cytoplasmic membranes. Purified glycerol-3-phosphate dehydrogenase was shown to donate electrons to ferricyanide (4), suggesting that this dehydrogenase is one of the ferricyanide sites. Detailed studies on another site(s) are planned.
DISCUSSION of Orientation membrane vesicles prepared by using a French press. D-LDH activity has been shown to be on the outside of the membrane vesicles prepared by using a French press. Thus all the markers tested so far, which are localized on the inside of the cytoplasmic membrane, were shown to be on the outside of the vesicles (3). The high rate of oxidative phosphorylation (9) and calcium transport (15) of these vesicles are consistent with the conclusion that they are inverted. The percentage of right-side-out vesicles in the preparation was estimated as less than 5%, by us (3) but as much as 20 to 40% by Altendorf and Staehelin (1). Our estimation was confirmed in this study by the fact that less than 10% of the D-lactate-dependent activity remained after addition of excess antibody. However, it must be noted that estimations by biochemical and morphological methods (1) are essentially different. Orientation of membrane vesicles prepared by treatment with EDTA-lysozyme. More than 85'% of the D-LDH in vesicles prepared by treatment with EDTA-lysozyme was inaccessible by anti-D-LDH. Our results seem essentially the same as those of Short et al. (19). However, unlike them we found that a small amount of activity (about 15%) was inhibited by antibody. This may represent the activity of completely inverted vesicles, like those prepared by using a French press, and the portion of completely inverted vesicles may vary in preparations made in different laboratories. Immunochemical studies do not provide information on the topology of this enzyme in the membrane and
J. BACTERIOL.
experiments are planned on this using another probe. We have shown that in these vesicles nearly half the activity of ferricyanide reductase, which is inside spheroplasts, is exposed (3). This was confirmed in the present study using membrane prepared by a modified procedure (19). Based on this observation we previously discussed the following possibilities (3): partial inversion of vesicles during their isolation, and movement of marker enzymes within the membrane vesicles during their preparation. However, inhibition of D-LDH in the membrane with the antibody would increase if 30 to 50% of the vesicles, in which ferricyanide reductase is detectable, are inverted. The most probable interpretation of the results is that the location of half the membrane protein which acts as ferricyanide sites changed during preparation of the vesicles, whereas most of the D-LDH (85%) molecules remained in their original location. The idea of movement of enzymes within the membrane during preparation of vesicles has already been suggested by Futai (3) and by Altendorf and Staehelin (1) as dislocation of membrane proteins. About half the ATPase in these vesicles was detectable (3, 21) and sensitive to its specific antibody (3). Short et al. (19) suggested that ATPase is dislocated, moving from the inner to the outer surface during homogenization with a Potter-Elvjem homogenizer. Recently, Hare et al. (7) fractionated membrane vesicles into inside-out and right-side-out vesicles using antibody against ATPase. Their preparation seemed to contain more inside-out vesicles than ours, because in our preparation only 15% of the D-LDH activity was inhibited by anti-D-LDH. Another possible explanation of our results is that inside-out vesicles in our preparation have D-LDH outside the membrane but that for some unknown reason it is not accessible to anti-DLDH. However, this explanation is inconsistent with the results of Short et al. (18) showing that the majority of the vesicles have transport activity and so are not inverted. Another possible argument is that immunochemical interaction of anti-r-LDH and o-LDH is somehow inhibited by contaminating outer membrane or lipopolysaccharide, although D-LDH is on the outside of the right-side-out vesicles which transport proline. This may be a minor possibility, as D-LDH activity in spheroplasts treated with toluene or in French press vesicles was sensitive to anti-r-LDH. However, for this control, it is desirable to have an antibody against an enzyme localizing outside the cyto-
ORIENTATION OF MEMBRANE VESICLES FROM E. COLI
VOL. 124, 1975
plasmic membrane. Interaction of such antibody and enzyme would be a perfect control for the above argument. However, we could not get such an enzyme and its antibody. Finally, the most probable interpretation of the results is as follows. (i) About 85% of vesicles in the preparation were right-side-out closed vesicles. About half of the ferricyanide reactive sites in EDTA-lysozyme vesicles were dislocated during isolation. (ii) About 15% of vesicles were inside-out (or unsealed membranes). ACKNOWLEDGMENTS We are grateful to E. P. Kennedy, H. R. Kaback, and L. D. Kohn for showing their manuscripts before publication and to L. A. Heppel for valuable information. We are also thankful to Y. Anraku for useful discussion. This work was partly supported by a grant from the Ministry of Education, Japan.
9.
10. 11. 12.
13.
us
LMTRATURE CITED 1. Altendorf, K. H., and L. A. Staehelin. 1974. Orientation of membrane vesicles from Escherichia coli, as detected by freeze-cleave electron microscopy. J. Bacteriol. 117:888-899. 2. Futai, M. 1973. Membrane D-lactate dehydrogenase from Escherichia coli. Purification and properties. Biochemistry 12:2468-2474. 3. Futai, M. 1974. Orientation of membrane vesicles from Escherichia coli prepared by different procedures. J. Membrane Biol. 15:15-28. 4. Futai, M. 1974. Reconstitution of transport dependent on D-lactate or glycerol-3-phosphate in membrane vesicles of Escherichia coli deficient in the corresponding dehydrogenase. Biochemistry 13:2327-2333. 5. Futai, M. 1974. Stimulation of transport into Escherichia
coli membrane vesicles by intemally generated reduced nicotinamide adenine dinucleotide. J. Bacteriol. 120:861-865. 6. Hampton, M. L., and E. Freese. 1974. Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles. J. Bacteriol. 118:497-504. 7. Hare, J. F., K. Olden, and E. P. Kennedy. 1974. Heterogeneity of membrane vesicles from Escherichia coli and their subfractionation with antibody to ATPase. Proc. Natl. Acad. Sci. U.S.A. 71:4843-4846. 8. Harold, F. M. 1972. Conservation and transformation of
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22.
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energy by bacterial membranes. Bacteriol. Rev. 36:172-230. Herzberg, E. M., and P. C. Hinkle. 1974. Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. Biochem. Biophys. Res. Commun. 58:178-184. Kaback, H. R. 1971. Bacterial membranes, p. 99-120. In W. B. Jacoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. Kaback, H. R. 1972. Transport across bacterial cytoplasmic membrane vesicles. Biochim. Biophys. Acta 265:367-416. Kohn, L. D., and H. R. Kaback. 1973. Mechanism of active transport in isolated bacterial membrane vesicles. XV. Purification and properties of the membrane bound D-lactate dehydrogenase from Escherichia coli. J. Biol. Chem. 248:7012-7017. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. McCauley, R., and E. Racker. 1973. Separation of two monoamine oxidase from bovine brain. Mol. Cell. Biochem. 1:73-81. Ouchtalony, 0. 1968. Handbook of immunodiffusion and immunology, p. 25. Arbor-Humphrey Science Publishers, Ann Arbor. Rosen, B. P., and J. S. McClees. 1974. Active transport of calcium in inverted membrane vesicles of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:5042-5046. Short, S. A., H. R. Kaback, T. Hawkins, and L. D. Kohn. 1975. Immunochemical properties of the membranebound D-lactate dehydrogenase from Escherichia coli. J. Biol. Chem. 250:4285-4290. Short, S. A., H. R. Kaback, G. Kaczorowski, J. Fisher, C. T. Walsh, and S. C. Silverstein. 1974. Determination of the absolute number of Escherichia coli membrane vesicles that catalyze active transport. Proc. Natl. Acad. Sci. U.S.A. 71:5032-5036. Short, S. A., H. R. Kaback, and L. D. Kohn. 1975. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J. Biol. Chem. 250:4291-4296. Tanaka, S., S. A. Lerner, and E. C. C. Lin. 1967. Replacement of a phosphoenol-pyruvate dependent phosphotransferase by a nicotinamide dinucleotidelinked dehydrogenase for the utilization of mannitol. J. Bacteriol. 93:642-648. van Thienen, G. and P. W. Postma. 1973. Coupling between energy conservation and active transport of serine in Escherichia coli. Biochim. Biophys. Acta 323:429-440. Weiner, J. H. 1974. The localization of glycerol-3-phosphate dehydrogenase in Escherichia coli. J. Membrane Biol. 15:1-14.
Vol. 124, No. 1 Printed in U.S.A.
Localization of D-Lactate Dehydrogenase in Membrane Vesicles Prepared by Using a French Press or Ethylenediaminetetraacetate-Lysozyme from Escherichia coli MASAMITSU FUTAI* AND YASUHITO TANAKA Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan Received for publication 29 May 1975
The localization of D-lactate dehydrogenase in membrane vesicles prepared from Escherichia coli was studied using antibody against the purified enzyme. The activity of D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by using a French press were completely inhibited by this antibody, suggesting that the enzyme is localized on the outside of these vesicles. This and previous results (Futai, 1974) strongly indicate the inversion of these vesicles. The D-lactate dehydrogenase and D-lactate-dependent oxygen uptake of membrane vesicles prepared by treatment with ethylenediaminetetraacetate-lysozyme were inhibited about 15% by the antibcody, whereas proline transport of the vesicles was insensitive to antibody. These results suggest that most of the membrane vesicles have D-lactate dehydrogenase on the inside of the membrane and that such vesicles transport amino acids. This essentially confirms the results of Short, Kaback, and Kohn (1975). However, unlike them we observed that a small but significant portion of activity was sensitive to the antibody as shown above. This portion may represent the completely inverted vesicles in the preparation. Ferricyanide reductase activity cannot be detected in spheroplasts, but about 30 to 50% of the total was detected in membrane vesicles prepared by treatment with ethylenediaminetetraacetate. This confirms our previous findings with membrane prepared by a slightly different procedure. It is concluded that in these vesicles about half the reactive sites for ferricyanide are moved from inside to outside the membrane, whereas 85% of the D-lactate dehydrogenase remains inside the membrane. Membrane vesicles prepared from Escherichia coli by using a French press (3, 9) and by treatment with ethylenediaminetetraacetate (EDTA)-lysozyme as described by Kaback (10) have been used for studies on oxidative phosphorylation (9) and active transport (11), respectively. We have suggested that membrane vesicles prepared by using a French press are inverted, as adenosine triphosphatase (ATPase), its binding sites, and ferricyanide reductase were found outside the membranes (3). Ferricyanide was formerly used as an impermeant electron acceptor from dehydrogenases (22). However, it is not a good electron acceptor for purified D-lactate dehydrogenase (4). This suggests that ferricyanide accepts electrons from a component(s) in the respiratory chain other than the dehydrogenase in the n-lactate oxidase system. Thus it seemed interesting to study the location of dehydrogenase itself in French press vesicles (membrane vesicles prepared by using a French press as described [31) by using immunochemical procedures.
Based on the above criteria we have also suggested that the orientation of half the membrane vesicles prepared by treatment with EDTA-lysozyme are changed from those of intact cells or spheroplasts (3). Evidence from other laboratories also suggests that EDTAlysozyme vesicles are a heterogeneous population (6, 7, 21). However, Kaback and his associates claimed that these vesicles are a homogenous right-side out population (10, 18, 19). Rosen and McClees demonstrated that EDTA-lysozyme vesicles did not transport calcium, whereas inverted French press vesicles do (16). Therefore, since EDTA-lysozyme vesicles are widely used for studies on transport, it is important to study the location of enzymes to determine the orientation of their membrane definitely. Topological information is also important for understanding the transport mechanism, as already pointed out by Harold (8). D-Lactate dehydrogenase (D-LDH), which is a membrane-bound primary dehydrogenase in
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ORIENTATION OF MEMBRANE VESICLES FROM E. COLI
471
the respiratory chain of E. coli, was solubilized Spheroplasts or membranes were treated with 1% and purified to homogeneity (2, 12). Recently toluene as described previously (3). The activity of was measured as phenazine methosulfate-couShort et al. reported that D-LDH in EDTA- D-LDHreduction of MTT (D-lactate:MTT reduction) lysozyme vesicles was not accessible by its pled (2). Ferricyanide reduction (3), proline transport (4), antibody, concluding that these vesicles are a oxygen uptake (4), and protein (13) were assayed as homogeneous right-side-out population (17, 19). described previously. Thus it is of interest to study the location of Materials. Pure i)-LDH was obtained as described D-LDH in EDTA-lysozyme vesicles prepared by previously (2). The chemicals used were as described us, as we have repeatedly observed their previously (2-4) or were the highest grade commercially available. heterogeneity (3, 22). In this paper the location of D-LDH in memRESULTS brane vesicles prepared from E. coli by using a anti-D-LDH. The antibody Properties of French press and by treatment with EDTAlysozyme was studied by using antibody against prepared against D-LDH was specific for memthe purified enzyme. Our results on EDTA- brane-bound D-LDH purified from E. coli ML lysozyme vesicles essentially agreed with Short 308-225. As shown in Fig. 1, on immunodiffuet al. (19), as a majority (85%) of the activity of sion a precipitin line was observed between D-lactate-dependent oxygen uptake and 3-(4,5dimethyl-2)2,5 diphenyl tetrazolium bromide (MTT) reduction was not inhibited by the antibody and none of the D-lactate-dependent proline uptake was inhibited. However, unlike them a small portion of activity (15%) of D-lactate-dependent oxygen uptake and D-lactate:MTT reduction was inhibited. These results are discussed in relation to the location of 6 ferricyanide reductase and to our previous findings (3, 22). MATERIALS AND METHODS Bacteria and growth conditions. E. coli ML308225 (i- z- y+ a+) was grown aerobically in synthetic medium (20) with 0.5% D,L-lactate as a carbon source, unless otherwise specified. 5 Preparation of membrane vesicles. Membrane
~2
K\)
4s
vesicles were prepared from EDTA-lysozyme spheroplasts as described by Kaback (10) using an 18-gauge needle for homogenization (19). Vesicles were also obtained by disruption of cells in a French press (pressure set, about 200 kg/cm2) (3). Both types of vesicles were used on the day of their preparation.
Preparation of antibody against I-LDH. Albino rabbits (about 1.5 kg) were immunized with 0.1 mg of purified D-LDH in complete Freund adjuvant (Difco Laboratories). Twenty days later they were injected with 0.05 mg of antigen in the same adjuvant. Thirty and 40 days after the second injection, they were injected with 0.05 mg of antigen in incomplete Freund adjuvant. For the injections, the antigen in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) containing 0.5% cholate was mixed with an equal volume of adjuvant. The mixture was injected subcutaneously. Animals were bled before immunization and 5 to 6 days after each booster injection. Antisera were examined by immunodiffusion (15). A precipitin line was usually detected after 4 or 5 booster injections, and gamma globulin was purified from the sera by ammonium sulfate precipitation and diethylaminoethyl-cellulose chromatography (13). Other procedures. Spheroplasts were prepared by using EDTA-lysozyme as described previously (3).
".
A",
-
i
FIG. 1. Immunodiffusion pattern of antiserum to D-LDH. Antiserum diluted fourfold with saline was placed in the center well and different proteins and detergents were placed in the peripheral wells. (1) Purified D-LDH (0.11 mg of protein/ml) in 0.05 M tris(hydroxymethyl)aminomethane (pH 7.5) containing 1.0%o Triton X-100. (2) Purified D-LDH (0.11 mg/ml) in the above buffer without Trition X-100. (3) Deoxycholate extract of membranes from ML 308-225 (4.5 mg/ml). (4) 1.0%o Triton X-100. (5) 1.0% cholate. (6) Supernatant solution obtained after lysis of spheroplasts in 0.07 M tris(hydroxymethyl)aminomethane buffer, pH 8.0 (26 mg/ml). About 5 Al of each sample was placed in the wells. Serum from preimmune rabbits did not form a precipitin line with purified D-LDH or deoxycholate extract (data not shown). After diffusion the plate was dried and stained with amido black.
472
J. BACTERIOL.
FUTAI AND TANAKA
anti-D-LDH and purified D-LDH or an extract of the membane with deoxycholate. No reaction was observed with the supernatant fraction obtained after lysis of the spheroplasts. Only one precipitin line was observed between anti-DLDH and membrane in 2% Triton X-100 in agarose containing the same concentration of Triton X-100 (data not shown). This suggests that the preparation of anti-D-LDH did not contain antibodies to other membrane proteins. Addition of anti-D-LDH to purified D-LDH caused time-dependent inhibition of activity; D-LDH was inhibited 90% on incubation with 200 ,ug of anti-D-LDH for 16 h (Fig. 2A). On the other hand, the r-globulin fraction obtained from rabbit before immunization had no effect on the activity and did not form a precipitin line with D-LDH in the immunodiffusion test. Excess anti-D-LDH (0.8 or 1.7 mg) had no effect on 1 U of L-LDH purified from E. coli ML 308-225 (dld-) (H. Kimura and M. Futai, manuscript in preparation), suggesting the specificity of the antibody. Effect of anti-D-LDH on the i-LDH activity in spheroplasts. As shown in Table 1, D-LDH activity in spheroplasts was not sensitive to anti-D-LDH when the spheroplasts were protected by sucrose. In this experiment, spheroplasts were preincubated for 1 h with anti-DLDH, as keeping them intact for more than 2 h was difficult even in the presence of sucrose. However, D-LDH activity became sensitive to the antibody when the spheroplasts were treated with toluene and preincubated with anti-D-LDH as above. On the other hand anti-DLDH had no effect on the activities of other dehydrogenases assayed in toluenized spheroplasts (see Table 1). These results suggest that D-LDH is localized on the inside of the cytoplasmic membrane and reduces the permeable dye used in the assay mixture. The activity of D-LDH in spheroplasts which had been treated with toluene was inhibited by the antibody less than that of purified enzyme or enzyme in membrane vesicles prepared by using a French press. This may be because the time of incubation was too short or the permeability of toluene-treated spheroplasts was too low. Effect of anti-z-LDH on membrane vesicles prepared by using a French press. Membranes were preincubated for 16 h in the following experiments, although substantial inhibition was observed even on incubation for 30 min. All the activities tested below were retained during preincubation in the absence of
antibody. Addition of anti-D-LDH to membrane vesicles prepared by using a French press caused
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400
800 400 800 0 () 400 pg ANTI D-LDH PER UNIT D- LDH
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FIG. 2. Effect of anti-D-LDH on purified D-LDH and membrane vesicles prepared by using a French press or treatment with EDTA-lysozyme. (A) Purified D-LDH (1.0 U) was incubated with different amounts of anti-D-LDH for 30 min (0) and 16 h (0) at 0 C in 0.2 ml of 0.05 M potassium phosphate buffer (pH 6.6) containing 0.23% Triton X-100, and D-lactate:MTT reduction was assayed. Activities are expressed as percentage of that of the control. (B) Membrane vesicles prepared by using a French press were incubated for 16 h at 0 C with different concentrations of anti-D-LDH in 0.22 ml of 0.05 Mpotassium phosphate buffer, pH 6.6. After incubation D-lactate:MTT reduction (A) and D-lactate-dependent oxygen uptake (0) were assayed and expressed as percentage of those of the controls (without antibody). The control rates of D-lactate:MTT reduction and D-lactate-dependent oxygen uptake were 0.28 Mmol and 108 ng of atom 0, respectively, per min per mg of protein. (As controls L-lactate, succinate, or glycerol3-phosphate dehydrogenase in French press vesicles were assayed under the same conditions. None of those activities was sensitive to anti-D-LDH up to 1.0 mg of protein/U of each enzyme. (C) Membrane vesicles prepared by treatment with EDTA-lysozyme as described by Kaback (10) and Short et al. (19) were incubated with anti-D-LDH in 0.05 M potassium phosphate buffer, pH 6.6, for 16 h at 0 C. After incubation D-lactate:MTT reductase (A), D-lactatedependent oxygen uptake (0), and proline uptake (U) were assayed. Activities are expressed as percentage of those of the control (without antibody). The control rates per minute per milligram of protein were: D-lac-
tate:MTT reductase, 0.45 ;tmol; D-lactate-dependent
oxygen uptake, 449 ng of atom 0; D-lactate-dependent proline uptake, 1.0 nmol.
almost complete inhibition of D-lactate-dependent reduction of MTT and oxygen uptake (Fig. 2B). D-Lactate-dependent reduction of ferricyanide was also inhibited to the same extent (data not shown). These results are in good agreement with our previous observations (3). Activities of other dehydrogenases in French press vesicles were not sensitive to anti-D-LDH, suggesting the inhibition observed above is specific (legend of Fig. 2B). Effects of anti-D-LDH on membrane vesi-
ORIENTATION OF MEMBRANE VESICLES FROM E. COLI
VOL. 124, 1975
TABLE 1. Effectofanti-D-LDHonD-LDHactivityin spheroplastsa D-LDH activity Anti-D-LDH
(4g) 0 50 100 200
(jAmol of MTT reduced/mg per min) Untreated
Treated with toluene
0.087 0.082 0.075 0.082
0.085 0.050 0.054 0.040
a Spheroplasts were obtained as described previously (3) except that 1 g (wet weight) of cells was suspended in 20 ml of a 20% sucrose solution. Spheroplasts (1.1 mg of protein) in 0.2 ml of 10% sucrose containing 0.01 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) were preincubated for 1 h at 20 C with different concentrations of anti-D-LDH. D-Lactate:MTT reduction was assayed in 10% sucrose using 10-MA aliquots of each preincubation mixture. Since spheroplast suspensions containing EDTA and lysozyme were directly used in the above assay, the concentration of cell protein was corrected for the amount of lysozyme added. One unit of L-lactate, succinate, or glycerol-3-phosphate dehydrogenase in spheroplasts treated with toluene was not inhibited by anti-D-LDH (up to 1.0 mg).
cles prepared by treatment with EDTA-lysozyme. As shown in Fig. 2C, D-lactate-dependent oxidation and D-lactate:MTT reduction in membrane vesicles prepared by treatment with EDTA-lysozyme were only slightly inhibited (about 15%) by the antibody. Furthermore, no inhibition of proline transport stimulated by D-lactate was observed. Short et al. (19) reported that neither of these activities were inhibited. The slight inhibition of D-lactate oxidation and D-lactate:MTT reduction we observed may be due to a difference in our preparation from theirs. Even with a large excess of antibody, less than 15% of the D-lactate oxidase activity and D-lactate:MTT reduction in this particular preparation were inhibited (Fig. 2C), although these activities were completely inhibited by antibody after sonic treatment of these vesicles (data not shown). We have repeated this titration with four different preparations of EDTA-lysozyme vesicles. The results suggested that the slight inhibitions of both activities observed by us are significant: (i) 400 and 800 ug of anti-D-LDH inhibited D-lactate-dependent oxygen uptake 15 i 5.3 and 14 :i 4.0%, respectively; (ii) 400 and 800 ug of anti-D-LDH inhibited D-lactate:MTT reduction 15 i 3.0 and 12 i 5.4%, respectively. D-Lactate-dependent uptake of proline by these different preparations was not sensitive to antiD-LDH. The interpretation of these results will be discussed below.
473
Ferricyanide reductase of membrane vesicles prepared by treatment with EDTAlysozyme. Ferricyanide reductase activity was not measurable in spheroplasts, but nearly half the total was detected in membrane vesicles prepared by EDTA-lysozyme using a PotterElvjem homogenizer (3, 22). Ferricyanide was suggested to be nonpermeable to the membranes of E. coli, since it inhibited transport into membrane vesicles stimulated by external reduced nicotinamide adenine dinucleotide, but not that stimulatd by internally generated reduced nicotinamide adenine dinucleotide (5). In this study membranes were dispersed with hypodermic needles instead of a homogenizer. This modification was made recently by Short et al. (19), who showed that it minimized the movement of ATPase from inside to outside the membrane during isolation of vesicles. Therefore, it seemed of interest to assay ferricyanide reduction by these membranes after preincubation in the presence or absence of toluene, which destroyed the permeability barrier for ferricyanide. As shown in Table 2, the activity of ferricyanide reductase was detected using glycerol3-phosphate, succinate, or D-lactate as substrate, and it increased about two- to threefold after the membranes had been treated with toluene. Toluene had no effect on the activity in membrane vesicles prepared by using a French TABLE 2. Ferricyanide reductase activity of membrane vesicles prepared by treatment with EDTA-lysozyme using a hypodermic needle for homogenizationa Ferricyanide reduction Substrate
(ismol reduced/mg per min) Untreated
Treated with toue toluene
Glycerol-3-
phosphate Succinate D-Lactate
0.049 0.017 0.026
0.140
0.034 0.056
aMembrane vesicles were prepared from E. coli ML 308-225 grown on 0.5% glycerol by the procedure of Kaback (10). A hypodermic needle was used to disperse membranes and spheroplasts throughout the preparation (19). Reduction of ferricyanide was followed spectrophotometrically at 420 nm in the presence of each substrate (20 mM). The increase of each activity of membrane vesicles prepared by using a French press could not be observed after toluene treatment. These results were confirmed by colorimetric assay using bathophenanthroline sulfonate (L. A. Heppel, personal communication). Other procedures were described in the text of a previous paper
(3).
474
FUTAI AND TANAKA
press, as shown previously (3). These results suggest that about 30 to 50% of ferricyanide sites are accessible, confirming our results on membranes prepared using a Potter-Elvjem homogenizer (3; L. A. Heppel confirmed this observation independently using membrane vesicles prepared from E. coli using a hypodermic needle for homogenization). It should be noted that this activity is not necessarily due to dehydrogenase, as discussed above, but it is provisionally assumed that it is due to a ferricyanide site(s), which is inaccessible from the outside of the cytoplasmic membranes. Purified glycerol-3-phosphate dehydrogenase was shown to donate electrons to ferricyanide (4), suggesting that this dehydrogenase is one of the ferricyanide sites. Detailed studies on another site(s) are planned.
DISCUSSION of Orientation membrane vesicles prepared by using a French press. D-LDH activity has been shown to be on the outside of the membrane vesicles prepared by using a French press. Thus all the markers tested so far, which are localized on the inside of the cytoplasmic membrane, were shown to be on the outside of the vesicles (3). The high rate of oxidative phosphorylation (9) and calcium transport (15) of these vesicles are consistent with the conclusion that they are inverted. The percentage of right-side-out vesicles in the preparation was estimated as less than 5%, by us (3) but as much as 20 to 40% by Altendorf and Staehelin (1). Our estimation was confirmed in this study by the fact that less than 10% of the D-lactate-dependent activity remained after addition of excess antibody. However, it must be noted that estimations by biochemical and morphological methods (1) are essentially different. Orientation of membrane vesicles prepared by treatment with EDTA-lysozyme. More than 85'% of the D-LDH in vesicles prepared by treatment with EDTA-lysozyme was inaccessible by anti-D-LDH. Our results seem essentially the same as those of Short et al. (19). However, unlike them we found that a small amount of activity (about 15%) was inhibited by antibody. This may represent the activity of completely inverted vesicles, like those prepared by using a French press, and the portion of completely inverted vesicles may vary in preparations made in different laboratories. Immunochemical studies do not provide information on the topology of this enzyme in the membrane and
J. BACTERIOL.
experiments are planned on this using another probe. We have shown that in these vesicles nearly half the activity of ferricyanide reductase, which is inside spheroplasts, is exposed (3). This was confirmed in the present study using membrane prepared by a modified procedure (19). Based on this observation we previously discussed the following possibilities (3): partial inversion of vesicles during their isolation, and movement of marker enzymes within the membrane vesicles during their preparation. However, inhibition of D-LDH in the membrane with the antibody would increase if 30 to 50% of the vesicles, in which ferricyanide reductase is detectable, are inverted. The most probable interpretation of the results is that the location of half the membrane protein which acts as ferricyanide sites changed during preparation of the vesicles, whereas most of the D-LDH (85%) molecules remained in their original location. The idea of movement of enzymes within the membrane during preparation of vesicles has already been suggested by Futai (3) and by Altendorf and Staehelin (1) as dislocation of membrane proteins. About half the ATPase in these vesicles was detectable (3, 21) and sensitive to its specific antibody (3). Short et al. (19) suggested that ATPase is dislocated, moving from the inner to the outer surface during homogenization with a Potter-Elvjem homogenizer. Recently, Hare et al. (7) fractionated membrane vesicles into inside-out and right-side-out vesicles using antibody against ATPase. Their preparation seemed to contain more inside-out vesicles than ours, because in our preparation only 15% of the D-LDH activity was inhibited by anti-D-LDH. Another possible explanation of our results is that inside-out vesicles in our preparation have D-LDH outside the membrane but that for some unknown reason it is not accessible to anti-DLDH. However, this explanation is inconsistent with the results of Short et al. (18) showing that the majority of the vesicles have transport activity and so are not inverted. Another possible argument is that immunochemical interaction of anti-r-LDH and o-LDH is somehow inhibited by contaminating outer membrane or lipopolysaccharide, although D-LDH is on the outside of the right-side-out vesicles which transport proline. This may be a minor possibility, as D-LDH activity in spheroplasts treated with toluene or in French press vesicles was sensitive to anti-r-LDH. However, for this control, it is desirable to have an antibody against an enzyme localizing outside the cyto-
ORIENTATION OF MEMBRANE VESICLES FROM E. COLI
VOL. 124, 1975
plasmic membrane. Interaction of such antibody and enzyme would be a perfect control for the above argument. However, we could not get such an enzyme and its antibody. Finally, the most probable interpretation of the results is as follows. (i) About 85% of vesicles in the preparation were right-side-out closed vesicles. About half of the ferricyanide reactive sites in EDTA-lysozyme vesicles were dislocated during isolation. (ii) About 15% of vesicles were inside-out (or unsealed membranes). ACKNOWLEDGMENTS We are grateful to E. P. Kennedy, H. R. Kaback, and L. D. Kohn for showing their manuscripts before publication and to L. A. Heppel for valuable information. We are also thankful to Y. Anraku for useful discussion. This work was partly supported by a grant from the Ministry of Education, Japan.
9.
10. 11. 12.
13.
us
LMTRATURE CITED 1. Altendorf, K. H., and L. A. Staehelin. 1974. Orientation of membrane vesicles from Escherichia coli, as detected by freeze-cleave electron microscopy. J. Bacteriol. 117:888-899. 2. Futai, M. 1973. Membrane D-lactate dehydrogenase from Escherichia coli. Purification and properties. Biochemistry 12:2468-2474. 3. Futai, M. 1974. Orientation of membrane vesicles from Escherichia coli prepared by different procedures. J. Membrane Biol. 15:15-28. 4. Futai, M. 1974. Reconstitution of transport dependent on D-lactate or glycerol-3-phosphate in membrane vesicles of Escherichia coli deficient in the corresponding dehydrogenase. Biochemistry 13:2327-2333. 5. Futai, M. 1974. Stimulation of transport into Escherichia
coli membrane vesicles by intemally generated reduced nicotinamide adenine dinucleotide. J. Bacteriol. 120:861-865. 6. Hampton, M. L., and E. Freese. 1974. Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles. J. Bacteriol. 118:497-504. 7. Hare, J. F., K. Olden, and E. P. Kennedy. 1974. Heterogeneity of membrane vesicles from Escherichia coli and their subfractionation with antibody to ATPase. Proc. Natl. Acad. Sci. U.S.A. 71:4843-4846. 8. Harold, F. M. 1972. Conservation and transformation of
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energy by bacterial membranes. Bacteriol. Rev. 36:172-230. Herzberg, E. M., and P. C. Hinkle. 1974. Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli. Biochem. Biophys. Res. Commun. 58:178-184. Kaback, H. R. 1971. Bacterial membranes, p. 99-120. In W. B. Jacoby (ed.), Methods in enzymology, vol. 22. Academic Press Inc., New York. Kaback, H. R. 1972. Transport across bacterial cytoplasmic membrane vesicles. Biochim. Biophys. Acta 265:367-416. Kohn, L. D., and H. R. Kaback. 1973. Mechanism of active transport in isolated bacterial membrane vesicles. XV. Purification and properties of the membrane bound D-lactate dehydrogenase from Escherichia coli. J. Biol. Chem. 248:7012-7017. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. McCauley, R., and E. Racker. 1973. Separation of two monoamine oxidase from bovine brain. Mol. Cell. Biochem. 1:73-81. Ouchtalony, 0. 1968. Handbook of immunodiffusion and immunology, p. 25. Arbor-Humphrey Science Publishers, Ann Arbor. Rosen, B. P., and J. S. McClees. 1974. Active transport of calcium in inverted membrane vesicles of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:5042-5046. Short, S. A., H. R. Kaback, T. Hawkins, and L. D. Kohn. 1975. Immunochemical properties of the membranebound D-lactate dehydrogenase from Escherichia coli. J. Biol. Chem. 250:4285-4290. Short, S. A., H. R. Kaback, G. Kaczorowski, J. Fisher, C. T. Walsh, and S. C. Silverstein. 1974. Determination of the absolute number of Escherichia coli membrane vesicles that catalyze active transport. Proc. Natl. Acad. Sci. U.S.A. 71:5032-5036. Short, S. A., H. R. Kaback, and L. D. Kohn. 1975. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J. Biol. Chem. 250:4291-4296. Tanaka, S., S. A. Lerner, and E. C. C. Lin. 1967. Replacement of a phosphoenol-pyruvate dependent phosphotransferase by a nicotinamide dinucleotidelinked dehydrogenase for the utilization of mannitol. J. Bacteriol. 93:642-648. van Thienen, G. and P. W. Postma. 1973. Coupling between energy conservation and active transport of serine in Escherichia coli. Biochim. Biophys. Acta 323:429-440. Weiner, J. H. 1974. The localization of glycerol-3-phosphate dehydrogenase in Escherichia coli. J. Membrane Biol. 15:1-14.
