ANALYTICAL
BIOCHEMISTRY
87,
91-96
(1978)
The Preparation of Tightly Coupled Membrane from Paracoccus denitrificans WRIGHT W. NICHOLS' Department
of Microbiology, Broad Street,
Vesicles
AND W. ALLAN HAMILTON
University Aberdeen
of Aberdeen, Marischal AB9 lAS, U. K.
College.
Received September 21, 1977; accepted January 3, 1978 Nonequilibrium sedimentation of membrane vesicles from Paracoccus through Ficoll gradients results in a separation into two fractions. The fraction which pellets through dense Ficoll is uncoupled. The second fraction, retarded by dense Ficoll, shows both improved concentrative transport activity and greater uncoupler stimulation of respiration as compared to the original vesicle preparation. denitrificans
Work in this laboratory has involved a study of the suitability of bacterial membrane vesicles for solving problems in microbial bioenergetics (1,2). We have been concerned with the orientation (3) and extent of energy coupling (1) of individual membrane vesicles within a particular population of vesicles. A standard method used for fractionating complex mixtures of membranes is density-gradient centrifugation in solutions of high molecular weight polymers (4). Therefore, we have studied the effect of centrifuging membrane vesicles from Paracoccus denitr$cans in density gradients composed of Ficoll. Density-gradient centrifugation of membrane vesicles from P. denitrificans does not separate the preparation into fractions of different membrane orientation. However, centrifugation in a Ficoll gradient, or indeed over a “pad” of dense Ficoll, causes the membrane vesicle population to divide into two fractions consisting of “uncoupled” membranes and “wellcoupled” membranes. The criteria used for this distinction are based on measurements of respiratory control (1) and active transport (25). METHODS Puracoccus denitrijcuns (NCIB 8944) was grown anaerobically at 30°C by the method of John and Whatley (6). One liter of medium contained: 10 ml of 0.5 M KHeP04, 10 ml of 0.5 M (NH,),HPO,, 0.1 ml of modified ’ Present address: Department of Biochemistry, Liverpool, L69 3BX, U. K. 91
University of Liverpool, P. 0. Box 147,
0003-2697178/0871-0091$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
92
NICHOLS
AND
HAMILTON
Hoagland trace element solution (7) 2 mg of ferric monosodium EDTA, 2 g of yeast extract 13.5 g of disodium succinate*6H,O, 10.1 g of KNO,. The pH was adjusted to 6.8 with H,SO,. Ca2+ and Mg2+ were autoclaved separately and added as 2.5 mg/ml CaC1,.2H,O (10 ml) and 2.5 mg/ml psS0,.7Hz0 (IO ml). Membrane vesicles were prepared as described in (2) and were used on the same day they were prepared. Continuous Ficoll density gradients were poured by the method of Britten and Roberts (8). The mixing chamber was stirred with a magnetic stirring bar and a vibrating glass rod. Centrifugation conditions are described in the legend to Fig. 1. Fractions of density gradients were taken from the top by piercing the base of the tube and the pellet, followed by pumping in 70% sucrose. The surface of the pellet was rinsed, and the pellet was resuspended in 10 mM Tris-acetate, pH 7.3. Oxygen uptake was assayed in a final volume of 3 ml at 30°C in a waterjacketed Clark-type oxygen electrode (Rank Bros., Cambridge). The vessel contained Tris-phosphate (10 mM), Mg acetate (1 mM), alcohol dehydrogenase (0.1 mg/ml), ethanol (I%, v/v), pH 7.3. The NADH-generating system was completed, after the addition of membrane vesicles, by the addition of 12 ~1 of NAD+ solution to a final concentration of 0.4 mM. Active transport of glycine was assayed at 30°C in the oxygen electrode vessel described above. This allowed monitoring of dissolved oxygen, which was maintained at (at least) 50% air-saturation level by bubbling in air from a microsyringe where necessary. Components of the reaction mixture were added as: 0.5 ml of Mg acetate (2 mM) in Tris-phosphate (20 mM), pH 7.3; membrane vesicles to a final protein concentration of approximately 2 mg/ml; water to bring the final volume to 0.9 ml. The experiment was initiated by the addition of 15 ~1 of [U-14C]glycine (5 &i/ pmol) to a final concentration of 15 PM. At the time indicated the electron donor, L-lactate, was added in 100 ~1 of aqueous solution to a final concen-
FIG. 1. The effect of sedimenting membrane vesicles fromP. denitrificans in a 2 to 5% Ficoll density gradient. The density gradient (final volume, 13 ml) contained 2 to 5% Ficoll, 0.5 mM ATP, 0.05 mM Mg acetate, and 0.5 mM Tris-acetate at pH 7.3. The gradient was loaded with 3 ml (30 mg of membrane protein) of vesicle suspension and centrifuged at 25,000 rpm (80,OOOg calculated at R,,) for 1 hr at 4°C. One-milliliter fractions (1- 11) were recovered as described in the Methods section.
ENERGY
COUPLING
IN BACTERIA
93
tration of 20 mM. Samples (50 ~1) were taken at times indicated and diluted in 0.1 M LiCl-1mM MgClz (2 ml) preequilibrated to 30°C. These diluted samples were immediately filtered under vacuum on a membrane filter assembly through Gelman GA8 filters (pore size, 0.2 pm; diameter, 25 mm). The filters were washed with 2 ml of the diluent, dried, and counted for radioactivity as described in Ref. (2). Protein was determined by the method of Lowry et al. (9) using bovine serum albumin as standard. [U-14C]glycine was obtained from the Radiochemical Centre, Amersham, Bucks. Ficoll was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. RESULTS
The result of one enrichment procedure used is shown in Fig. 1. A major portion of membrane vesicles from P. d~nitrifcans is retarded by dense Ficoll and bands (after 1 hr at 80,OOOg) between 3 and 4% Ficoll. The remainder of the vesicles are pelleted in the gradient shown in Fig. 1; i.e., they pass through a 5% Ficoll solution under the described centrifugation conditions. We note, however, that membrane vesicles are distributed at all densities below 3% Ficoll. This is concluded from the light-red color in that part of the gradient and from the demonstration of coupled NADH oxidase activity in Fractions 4 through 9 (Table 1). Two further observations are noteworthy. The Ficoll solution in the centrifuge tube need not be present as a gradient. Layering membrane vesicles over a simple “pad” of 10% Ficoll also results in the separation into two populations under similar centrifugation conditions. About 40 to 60% of the vesicles do not enter 10% Ficoll (these behave like the retarded vesicles in Fig. I), the remainder are pelleted (these behave like the pelleted vesicles in Fig. 1). Secondly, these effects are not observed with membrane vesicles which have been frozen in liquid nitrogen, our principle method of storage (2). The whole of a freeze-thaw-cycled preparation is pelleted through a pad of 10% Ficoll. Table 1 shows the respiratory activities of the individual fractions obtained from the gradient depicted in Fig. 1. The pellet is poorly coupled, shown by its uncoupler stimulation of 1.6 (as compared with 4.8 for the original preparation). The fractions comprising the membrane vesicles retained in the gradient (principally Fractions 10 and 11) have significantly higher uncoupler stimulations than the original preparation. Enrichment of coupled vesicles is also observed when active transport is assayed (Fig. 2). Vesicles retarded by the dense Ficoll have an increased rate of respiration-dependent glycine transport than the original preparation (an initial rate of 0.15 nmol of glycine min-’ mg-’ of protein as opposed to 0.07 nmol of glycine mine1 mg-’ of protein). Conversely, the pellet has
94
NICHOLS
AND HAMILTON TABLE
NADH
OXIDASE
ACTIVITIES
OBTAINED
FROM
AND
UNCOUPLER
THE
DENSITY
I STIMULATIONS
GRADIENT
SHOWN
OBSERVED
IN FRACTIONS
IN FIGURE
1”
NADH oxidase rates (ng atom of Oxygen min-’ one-milliliter fraction-‘)
Original preparation Resuspended pellet Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Fraction 10 Fraction 11
Basal rate
Gramicidin and NH,+
Ratio of uncoupled/ control respiration rates
4,840 8,100 -fJ 470 350 540 570 560 280 710 400
23.300 13,100 2,060 1,720 2,060 2,900 3,350 1,760 4.290 4,580
4.8 1.6 4.4 4.9 3.8 5.1 6.0 6.3 6.0 11.4
n NADH oxidase activities were assayed as described in the Methods section. Gramicidin was added as 3 ~1 acetone solution to 0.3 &ml and NH,+ as IO ~1 aqueous solution to 10 mM. Acetone alone had no effect. b Not tested.
very low concentrative of protein).
transport activity (0.02 nmol of glycine min-’ mg-’
DISCUSSION
Extent of Coupling The first demonstration of bacterial respiratory control, as defined by Chance and Williams (lo), was achieved by John and Hamilton (I), who demonstrated the ADP-dependent increase in NADH oxidase activity of membrane vesicles from P. denitri~cans. In addition, the NADH oxidase activity of these membrane vesicles may also be stimulated by uncouplers, particularly gramicidin in the presence of NH,+ [Table 1; Ref. (3)]. It is therefore possible to quantitate the degree of coupling of these respiratory membrane vesicles as either the true respiratory control ratio (1) or, as we have done in Table 1, an uncoupled/control respiratory ratio. We have observed that membrane vesicles prepared from P. dentrificans by our procedure catalyze active transport (2). Figure 2 shows that the transport of glycine is active transport since it is dependent on the
ENERGY COUPLING
IN BACTERIA
95
FIG. 2. The concentrative transport of glycine by membrane vesicles from P. denitri$cans fractionated by centrifugation in a Ficoll density gradient: (0) control, original vesicle preparation; (A) combined Fractions 4 to 11 from the Ficoll density gradient; (0) resuspended pellet from the Ficoll density gradient. Fractions 4 to 11 from Fig. 1 (NADH oxidase activities shown in Table 1) were combined and pelleted by centrifugation at 18,000 rpm (40,OOOg calculated at R,,,) for 40 min at 4°C. The membrane vesicles were resuspended to 6.3 mg of protein/ml in 10 mM tris-acetate, pH 7.3. The pellet from Fig. 1 was resuspended in the same buffer to 9.3 mg of protein/ml. Concentrative glycine transport was measured as described in the Methods section. The arrow indicates the addition of L-lactate (20 mM).
addition of electron donor. Furthermore, glycine is discharged by the addition of the uncoupler gramicidin plus ammonium ions (not shown). We must stress, therefore, that the membrane vesicle preparations used by us are mixtures of inside-out vesicles, since they dispay ADP-linked respiratory control [Fig. 1; Ref. (3)], and right-side-out vesicles, since they possess the capacity for concentrative transport (Fig. 2). This problem has been pointed out and investigated in detail by Burnell et al. (11). Enrichment
of Coupled
Vesicles
From Fig. 1 we see that only a proportion of membrane vesicles from P. dentrifcans pass right through a 2 to 5% Ficoll gradient. The remainder are retained at about 4% Ficoll under the centrifugation conditions employed. In other experiments utilizing gradients of 10 to 30% Ficoll or a pad of 10% Ficoll, equivalent bands of vesicles are observed. We therefore have a rapid and simple method for significantly enriching membrane vesicle preparations with regard to coupled vesicles of insideout or right-side-out orientation. Furthermore, we suggest that the basis of the separation between coupled and uncoupled vesicles (regardless of their orientation) rests on their permeability; i.e., uncoupled vesicles areunsealed and travel through dense Ficoll under our centrifugation con-
96
NICHOLS
AND HAMILTON
ditions. Coupled vesicles, however, are resealed and are impermeable to Ficoll and therefore remain in the less-dense Ficoll layers under these centrifugation conditions. Kant and Steck (12) have drawn identical conclusions concerning the behavior in dextran density gradients of sealed and unsealed membrane vesicles (again regardless of orientation) from human erythrocytes. This strongly supports our interpretation of the presented data. ACKNOWLEDGMENT WWN gratefully acknowledges receipt of a Medical Research Council Scholarship for Training in Research Methods.
REFERENCES 1. 2. 3. 4.
John, P., and Hamilton, W. A. Nichols, W. W., and Hamilton, John, P., and Hamilton, W. A. Steck, T. L., Straus, J. H., and
(1970) FEBS Lett. 10, 246-248. W. A. (1976) FEBS Left. 65, 107-110. (1971) Eur. J. Biochem. 23, 528-532. Wallach, D. F. H. (1970) Biochim. Biophys. Acra 203,
385-393.
5. White, D. C., Tucker, D. N., and Kaback, H. R. (1974) Arch. Biochem. Biophys. 165, 672-680.
John, P., and Whatley, F. R. (1970) Biochim. Biophys. Acta 216, 342-352. Collins, V. G. (1%9)in Methods in Microbiology (Norris, J. R., and Ribbons, D. W., eds.) Vol. 3B, pp. l-52, Academic Press, New York. 8. Britten, R. J., and Roberts, R. B. (1960) Science 131, 32-33. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 6. 7.
193, 265-275.
10. Chance, B., and Williams, G. R. (1955)J. Biof. Chem. 217, 383-407. 11. Burnell, J. N., John, P., and Whatley, F. R. (1975) B&hem. J. 150, 527-536. 12. Kant, J. A., and Steck, T. L. (1972) Nature (London) 240, 26-28.
BIOCHEMISTRY
87,
91-96
(1978)
The Preparation of Tightly Coupled Membrane from Paracoccus denitrificans WRIGHT W. NICHOLS' Department
of Microbiology, Broad Street,
Vesicles
AND W. ALLAN HAMILTON
University Aberdeen
of Aberdeen, Marischal AB9 lAS, U. K.
College.
Received September 21, 1977; accepted January 3, 1978 Nonequilibrium sedimentation of membrane vesicles from Paracoccus through Ficoll gradients results in a separation into two fractions. The fraction which pellets through dense Ficoll is uncoupled. The second fraction, retarded by dense Ficoll, shows both improved concentrative transport activity and greater uncoupler stimulation of respiration as compared to the original vesicle preparation. denitrificans
Work in this laboratory has involved a study of the suitability of bacterial membrane vesicles for solving problems in microbial bioenergetics (1,2). We have been concerned with the orientation (3) and extent of energy coupling (1) of individual membrane vesicles within a particular population of vesicles. A standard method used for fractionating complex mixtures of membranes is density-gradient centrifugation in solutions of high molecular weight polymers (4). Therefore, we have studied the effect of centrifuging membrane vesicles from Paracoccus denitr$cans in density gradients composed of Ficoll. Density-gradient centrifugation of membrane vesicles from P. denitrificans does not separate the preparation into fractions of different membrane orientation. However, centrifugation in a Ficoll gradient, or indeed over a “pad” of dense Ficoll, causes the membrane vesicle population to divide into two fractions consisting of “uncoupled” membranes and “wellcoupled” membranes. The criteria used for this distinction are based on measurements of respiratory control (1) and active transport (25). METHODS Puracoccus denitrijcuns (NCIB 8944) was grown anaerobically at 30°C by the method of John and Whatley (6). One liter of medium contained: 10 ml of 0.5 M KHeP04, 10 ml of 0.5 M (NH,),HPO,, 0.1 ml of modified ’ Present address: Department of Biochemistry, Liverpool, L69 3BX, U. K. 91
University of Liverpool, P. 0. Box 147,
0003-2697178/0871-0091$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
92
NICHOLS
AND
HAMILTON
Hoagland trace element solution (7) 2 mg of ferric monosodium EDTA, 2 g of yeast extract 13.5 g of disodium succinate*6H,O, 10.1 g of KNO,. The pH was adjusted to 6.8 with H,SO,. Ca2+ and Mg2+ were autoclaved separately and added as 2.5 mg/ml CaC1,.2H,O (10 ml) and 2.5 mg/ml psS0,.7Hz0 (IO ml). Membrane vesicles were prepared as described in (2) and were used on the same day they were prepared. Continuous Ficoll density gradients were poured by the method of Britten and Roberts (8). The mixing chamber was stirred with a magnetic stirring bar and a vibrating glass rod. Centrifugation conditions are described in the legend to Fig. 1. Fractions of density gradients were taken from the top by piercing the base of the tube and the pellet, followed by pumping in 70% sucrose. The surface of the pellet was rinsed, and the pellet was resuspended in 10 mM Tris-acetate, pH 7.3. Oxygen uptake was assayed in a final volume of 3 ml at 30°C in a waterjacketed Clark-type oxygen electrode (Rank Bros., Cambridge). The vessel contained Tris-phosphate (10 mM), Mg acetate (1 mM), alcohol dehydrogenase (0.1 mg/ml), ethanol (I%, v/v), pH 7.3. The NADH-generating system was completed, after the addition of membrane vesicles, by the addition of 12 ~1 of NAD+ solution to a final concentration of 0.4 mM. Active transport of glycine was assayed at 30°C in the oxygen electrode vessel described above. This allowed monitoring of dissolved oxygen, which was maintained at (at least) 50% air-saturation level by bubbling in air from a microsyringe where necessary. Components of the reaction mixture were added as: 0.5 ml of Mg acetate (2 mM) in Tris-phosphate (20 mM), pH 7.3; membrane vesicles to a final protein concentration of approximately 2 mg/ml; water to bring the final volume to 0.9 ml. The experiment was initiated by the addition of 15 ~1 of [U-14C]glycine (5 &i/ pmol) to a final concentration of 15 PM. At the time indicated the electron donor, L-lactate, was added in 100 ~1 of aqueous solution to a final concen-
FIG. 1. The effect of sedimenting membrane vesicles fromP. denitrificans in a 2 to 5% Ficoll density gradient. The density gradient (final volume, 13 ml) contained 2 to 5% Ficoll, 0.5 mM ATP, 0.05 mM Mg acetate, and 0.5 mM Tris-acetate at pH 7.3. The gradient was loaded with 3 ml (30 mg of membrane protein) of vesicle suspension and centrifuged at 25,000 rpm (80,OOOg calculated at R,,) for 1 hr at 4°C. One-milliliter fractions (1- 11) were recovered as described in the Methods section.
ENERGY
COUPLING
IN BACTERIA
93
tration of 20 mM. Samples (50 ~1) were taken at times indicated and diluted in 0.1 M LiCl-1mM MgClz (2 ml) preequilibrated to 30°C. These diluted samples were immediately filtered under vacuum on a membrane filter assembly through Gelman GA8 filters (pore size, 0.2 pm; diameter, 25 mm). The filters were washed with 2 ml of the diluent, dried, and counted for radioactivity as described in Ref. (2). Protein was determined by the method of Lowry et al. (9) using bovine serum albumin as standard. [U-14C]glycine was obtained from the Radiochemical Centre, Amersham, Bucks. Ficoll was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. RESULTS
The result of one enrichment procedure used is shown in Fig. 1. A major portion of membrane vesicles from P. d~nitrifcans is retarded by dense Ficoll and bands (after 1 hr at 80,OOOg) between 3 and 4% Ficoll. The remainder of the vesicles are pelleted in the gradient shown in Fig. 1; i.e., they pass through a 5% Ficoll solution under the described centrifugation conditions. We note, however, that membrane vesicles are distributed at all densities below 3% Ficoll. This is concluded from the light-red color in that part of the gradient and from the demonstration of coupled NADH oxidase activity in Fractions 4 through 9 (Table 1). Two further observations are noteworthy. The Ficoll solution in the centrifuge tube need not be present as a gradient. Layering membrane vesicles over a simple “pad” of 10% Ficoll also results in the separation into two populations under similar centrifugation conditions. About 40 to 60% of the vesicles do not enter 10% Ficoll (these behave like the retarded vesicles in Fig. I), the remainder are pelleted (these behave like the pelleted vesicles in Fig. 1). Secondly, these effects are not observed with membrane vesicles which have been frozen in liquid nitrogen, our principle method of storage (2). The whole of a freeze-thaw-cycled preparation is pelleted through a pad of 10% Ficoll. Table 1 shows the respiratory activities of the individual fractions obtained from the gradient depicted in Fig. 1. The pellet is poorly coupled, shown by its uncoupler stimulation of 1.6 (as compared with 4.8 for the original preparation). The fractions comprising the membrane vesicles retained in the gradient (principally Fractions 10 and 11) have significantly higher uncoupler stimulations than the original preparation. Enrichment of coupled vesicles is also observed when active transport is assayed (Fig. 2). Vesicles retarded by the dense Ficoll have an increased rate of respiration-dependent glycine transport than the original preparation (an initial rate of 0.15 nmol of glycine min-’ mg-’ of protein as opposed to 0.07 nmol of glycine mine1 mg-’ of protein). Conversely, the pellet has
94
NICHOLS
AND HAMILTON TABLE
NADH
OXIDASE
ACTIVITIES
OBTAINED
FROM
AND
UNCOUPLER
THE
DENSITY
I STIMULATIONS
GRADIENT
SHOWN
OBSERVED
IN FRACTIONS
IN FIGURE
1”
NADH oxidase rates (ng atom of Oxygen min-’ one-milliliter fraction-‘)
Original preparation Resuspended pellet Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Fraction 10 Fraction 11
Basal rate
Gramicidin and NH,+
Ratio of uncoupled/ control respiration rates
4,840 8,100 -fJ 470 350 540 570 560 280 710 400
23.300 13,100 2,060 1,720 2,060 2,900 3,350 1,760 4.290 4,580
4.8 1.6 4.4 4.9 3.8 5.1 6.0 6.3 6.0 11.4
n NADH oxidase activities were assayed as described in the Methods section. Gramicidin was added as 3 ~1 acetone solution to 0.3 &ml and NH,+ as IO ~1 aqueous solution to 10 mM. Acetone alone had no effect. b Not tested.
very low concentrative of protein).
transport activity (0.02 nmol of glycine min-’ mg-’
DISCUSSION
Extent of Coupling The first demonstration of bacterial respiratory control, as defined by Chance and Williams (lo), was achieved by John and Hamilton (I), who demonstrated the ADP-dependent increase in NADH oxidase activity of membrane vesicles from P. denitri~cans. In addition, the NADH oxidase activity of these membrane vesicles may also be stimulated by uncouplers, particularly gramicidin in the presence of NH,+ [Table 1; Ref. (3)]. It is therefore possible to quantitate the degree of coupling of these respiratory membrane vesicles as either the true respiratory control ratio (1) or, as we have done in Table 1, an uncoupled/control respiratory ratio. We have observed that membrane vesicles prepared from P. dentrificans by our procedure catalyze active transport (2). Figure 2 shows that the transport of glycine is active transport since it is dependent on the
ENERGY COUPLING
IN BACTERIA
95
FIG. 2. The concentrative transport of glycine by membrane vesicles from P. denitri$cans fractionated by centrifugation in a Ficoll density gradient: (0) control, original vesicle preparation; (A) combined Fractions 4 to 11 from the Ficoll density gradient; (0) resuspended pellet from the Ficoll density gradient. Fractions 4 to 11 from Fig. 1 (NADH oxidase activities shown in Table 1) were combined and pelleted by centrifugation at 18,000 rpm (40,OOOg calculated at R,,,) for 40 min at 4°C. The membrane vesicles were resuspended to 6.3 mg of protein/ml in 10 mM tris-acetate, pH 7.3. The pellet from Fig. 1 was resuspended in the same buffer to 9.3 mg of protein/ml. Concentrative glycine transport was measured as described in the Methods section. The arrow indicates the addition of L-lactate (20 mM).
addition of electron donor. Furthermore, glycine is discharged by the addition of the uncoupler gramicidin plus ammonium ions (not shown). We must stress, therefore, that the membrane vesicle preparations used by us are mixtures of inside-out vesicles, since they dispay ADP-linked respiratory control [Fig. 1; Ref. (3)], and right-side-out vesicles, since they possess the capacity for concentrative transport (Fig. 2). This problem has been pointed out and investigated in detail by Burnell et al. (11). Enrichment
of Coupled
Vesicles
From Fig. 1 we see that only a proportion of membrane vesicles from P. dentrifcans pass right through a 2 to 5% Ficoll gradient. The remainder are retained at about 4% Ficoll under the centrifugation conditions employed. In other experiments utilizing gradients of 10 to 30% Ficoll or a pad of 10% Ficoll, equivalent bands of vesicles are observed. We therefore have a rapid and simple method for significantly enriching membrane vesicle preparations with regard to coupled vesicles of insideout or right-side-out orientation. Furthermore, we suggest that the basis of the separation between coupled and uncoupled vesicles (regardless of their orientation) rests on their permeability; i.e., uncoupled vesicles areunsealed and travel through dense Ficoll under our centrifugation con-
96
NICHOLS
AND HAMILTON
ditions. Coupled vesicles, however, are resealed and are impermeable to Ficoll and therefore remain in the less-dense Ficoll layers under these centrifugation conditions. Kant and Steck (12) have drawn identical conclusions concerning the behavior in dextran density gradients of sealed and unsealed membrane vesicles (again regardless of orientation) from human erythrocytes. This strongly supports our interpretation of the presented data. ACKNOWLEDGMENT WWN gratefully acknowledges receipt of a Medical Research Council Scholarship for Training in Research Methods.
REFERENCES 1. 2. 3. 4.
John, P., and Hamilton, W. A. Nichols, W. W., and Hamilton, John, P., and Hamilton, W. A. Steck, T. L., Straus, J. H., and
(1970) FEBS Lett. 10, 246-248. W. A. (1976) FEBS Left. 65, 107-110. (1971) Eur. J. Biochem. 23, 528-532. Wallach, D. F. H. (1970) Biochim. Biophys. Acra 203,
385-393.
5. White, D. C., Tucker, D. N., and Kaback, H. R. (1974) Arch. Biochem. Biophys. 165, 672-680.
John, P., and Whatley, F. R. (1970) Biochim. Biophys. Acta 216, 342-352. Collins, V. G. (1%9)in Methods in Microbiology (Norris, J. R., and Ribbons, D. W., eds.) Vol. 3B, pp. l-52, Academic Press, New York. 8. Britten, R. J., and Roberts, R. B. (1960) Science 131, 32-33. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 6. 7.
193, 265-275.
10. Chance, B., and Williams, G. R. (1955)J. Biof. Chem. 217, 383-407. 11. Burnell, J. N., John, P., and Whatley, F. R. (1975) B&hem. J. 150, 527-536. 12. Kant, J. A., and Steck, T. L. (1972) Nature (London) 240, 26-28.
