SEDIMENTATION STUDIES ON MEMBRANE VESICLES
J. BOOM, W.S. BONT, H.P. HOFS, and M. DE VRIES Division of Biophysics, Antoni van Leeuwenhoek-Huis, The Netherlands Cancer Institute, Sarphatistraat 108, Amsterdam, The Netherlands
(Received 27 May, 1976) Abstract. Analytical centrifugation was used in order to investigate the size distribution of vesicles in various membrane preparations. Under certain conditions depending among others on the speed of rotation and the temperature, a sedimentation profile was observed that was characteristic for membraneous material. Since the membrane vesicles exhibited a discontinuous distribution of sedimentation coefficients it was concluded that membrane vesicles only occur in a few size classes.
I. INTRODUCTION Membranes from various parts of the cell are characterized by their chemical composition, enzymatic activities and buoyant density. When large membraneous structures are isolated, fragmentation of the membranes is unavoidable. The morphology of these fragmented membranes has been studied with the electron microscope. The finding that vesicles are often formed appears to be related to the fact that bilayers formed by hydrophobic lipids are the major constituents of biomembranes [ 1]. To our knowledge no systematic investigation concerning the distribution of vesicle sizes in preparations of biomembranes has been made.
II. MATERIALS AND METHODS
1. The Preparation o f Nuclear Membranes During the whole procedure a temperature of 2 - 4 ~ was maintained. The liver of a 3-monthsold male rat from the inbred strain R-Amsterdam was forced through a metal gauze with pores of 1 mm 2, and homogenized in 10 vol of a medium containing 0.10 M NaCI, 0.015 M EDTA, 0.75 M sucrose, 0.04 M Tris HCI, pH 7.5 (medium 2) using a teflon homogenizer of the PotterElvehjem type with a clearance of 0.5 mm during about 60 s with a velocity of 1400 rev min-1. The homogenate was centrifuged for 5 min at 2000 X g and the sediment gently resuspended in 10 vol of a medium containing 0.10 M NaC1, 0.014 M EDTA, 0.04 M Tris HC1, pH 7.5 (medium 1) (cf. [2]) using the same homogenizer driven by hand. After 20 min stirring in the cold room we centrifuged for 5 min at 2000 • g; this step was repeated once. Although the sediment was 81 Molecular Biology Reports 3 (1976) 81-86. All Rights Reserved. Copyright 9 1976 by D. Reidel Publishing Company, Dordrecht-Holland.
not free from cytoplasmic contaminants as could be seen by phasecontrast microscopy, this crude preparation of nuclei was routinely used for the extraction of membranes. The wall of the centrifuge tube was rinsed and the sediment washed with a small volume of bidistilled water. After centrifugation for 5 min at 2000 X g the sediment was carefully resuspended in 25 ml bidistilled water per liver, with the aid of a hand-driven teflon homogenizer having a wide gap between pestle and mortar (clearance approx. 1 mm). This suspension was introduced in a pressure homogenizer (Baskerville and Lindsay Ltd., Manchester) as described by Hanter and Commerford [3]. Nitrogen was slowly admitted at a rate such that it took about 5 min to reach a pressure of 800 lb inch -2. After 15 min the suspension was expelled from the pressure vessel and centrifuged for 15 min at 10 000 X g. The sediment was discarded and the supernatant was layered on 10 ml of a 2 M sucrose solution in a tube, fitted for the type 30 rotor of the Spinco Model L preparative ultracentrifuge. The tube was equilibrated with bidistilled water, and centrifuged for 30 min at 100 000 • g. The material in the interphase was collected and used for further investigations.
2. Preparation of Membranes of the Endoplasmic Reticulum Both smooth and rough membranes of the endoplasmic reticulum were isolated from rat liver cells as described previously [4].
3. Analytical Centrifugation The preparations were analyzed at 260 nm in a Spinco Model E analytical ultracentrifuge equipped with UV absorption optics and an automatic scanning system developed in this laboratory [5]. The concentrations varied from about 0.5 mg m1-1 for membranes from the endoplasmic reticulum to 0.1 mg ml-1 for plasma membranes for analysis in the ultracentrifuge. The absorbance of all preparations was between 1 and 2 units.
III. RESULTS
1. Sedimentation Analysis of Nuclear Membranes The sedimentation velocity was studied in a Spinco Model E analytical ultracentrifuge equipped with U.V. absorption optics coupled with an automatic scanning system as described previously [5]. The results of a typical sedimentation experiment are given in Figure 1. It is evident that the membrane contained well defined fractions as revealed by the steplike sedimentation pattern. The sedimentation coefficients of fractions i-7 (see Figure 1) were 131 S, 251 S, 425 S, 610 S, 869 S, 1169 S and 2163 S, respectively.
2. Sedimentation Analysis of Cytoplasmic Membranes We were interested to know whether these findings were restricted to membranes isolated from a nuclear fraction or that it was an aspect of biomembranes in general. One could argue that nuclear membranes contain DNA, be it only as a small fraction of the total mass of the membraneous material. This non-membraneous nuclear material could possibly be responsible for the peculiar fragmentation of the nuclear membranes. Therefore we decided to investigate the membranes of the endoplasmic reticulum. It is well known that the membrane fraction of 82
Fig. 1. Sedimentation analysis of nuclear membranes. The membranes were isolated as described under 'Materials and Methods' and diluted with bidistilled water to an absorbance at 260 nm of about 1.5. The sedimentation prof'des were traced at 1.5 min, 4.5 min, 13.5 min and 19.5 min, respectively, after reaching maximum speed (= 16 200 rev min-l). Temperature = 20 ~ M = meniscus at a radial distance of 5.88 cm. For the sedimentation coefficients of the 7 components see text.
the endoplasmic reticulum consists o f two groups o f membranes viz the rough membranes, i.e. membranes dotted with ribosomes, and smooth membranes, i.e. membranes without ribosomes [6]. Both smooth membranes and rough membranes were analyzed and in Figure 2 a typical sedimentation profile obtained for smooth membranes is given. The 6 components had S-values o f 150 S, 425 S, 778 S, 1016 S, 1539 S and 3653 S, respectively.
IV. DISCUSSION Membrane preparations that exist in the form of vesicles were examined in the analytical ultracentrifuge equipped with absorption optics. When analyzing the tracings, we have to keep in mind that the absorbance o f membraneous material at 260 # m was due to light-scattering, since the absorbance diminished after the addition o f a detergent, e.g. SDS, to the suspension. By the addition o f detergents membrane-vesicles are broken down into particles too small to give 83
Fig. 2. Sedimentation analysis of membranes of the endoplasmic reticulum. The membranes were isolated as described under 'Materials and Methods' and diluted to absorbance at 260 nm of about 1.5, with a medium containing 0.07 M KC1, 0.05 M Tris pH 7.6 and 8 mM MgCI.The sedimentation profiles were traced at 0 min, 3 rain and 7.5 min, respectively, after reaching maximum speed (-- 13 600 rev min-1). Temperature = 20 ~C. M = radial distance of meniscus. R = reference point at 7.3 cm. For the sedimentation coefficients of the 6 components see text. light-scattering at 260 nm [7]. Since the rough membranes o f the endoplasmic reticulum were heavily contaminated with ribosomes, part of the absorbance at 260 nm must be ascribed to the ribosomes and this in turn explains why the concentration of material corresponding to about 1 absorption unit in these preparations was higher (0.5 mg ml-1) than in the membrane preparations derived from the nucleus and the plasma membrane (0.1 mg ml-1). When complex structures like biomembranes are fragmented, one would expect a random distribution o f sizes with a mean size dependent on the mode o f preparation o f the membraneous material. With very subtle isolation methods the mean size should correspond to very large particles; a crude isolation procedure should result in relatively small particles. The fact that fragments of nuclear membranes only exist in a few classes, that are characterized by well 84
defined sedimentation coefficients, poses the question whether this is a more general phenomenon of biomembranes. In order to answer this question the vesicles of membranes were separated on sucrose gradients. The heavier fractions contained only components with high S-values, the light fractions only low S-values, and the intermediate fractions almost exclusively the intermediate S-values. In addition the S-values could be correlated with the results obtained with the electronmicroscope: the higher the S-value the larger the particles. It was therefore concluded that the sedimentation profde was not due to sedimentation artifacts. Each ~sedimenting component consists of a vesicle with definite mass. The more rapidly sedimenting components are not aggregates of smaller ones since electronmicroscopic studies revealed that the heavier components did not contain aggregates of small vesicles. Now one could argue that biomembranes have a mosaic structure and when the membranes are disintegrated, rupture leaves certain fragments of the mosaic intact. A component with a definite sedimentation coefficient should then correspond to a certain part of the mosaic. However, artificial membranes (liposomes) also exhibited a discontinuous size distribution and this again favours the assumption that the different vesicles do not represent various identical structures of a mosaic present in biomembranes. It was concluded therefore that vesicles of biomembranes occur in only a few size classes. The finding that certain cellular components, as e.g. excretion granulae, are homogeneous in size [8] might indicate that also in the intact biomembranes a purely physical phenomenon is operative for the determination of particle size. When the S-values for the microsomal membranes and the nuclear membranes were compared it was striking that certain components in the nuclear membranes had the same S-value as those in the microsomal membranes. Thus components 1, 2, 3 and 4 of the microsomal membranes had approximately the same S-value (less than 15% difference) as components 1, 3, 5 and 6, respectively, of the nuclear membranes. Only two of the many experiments (about 20 for each type of preparation) were shown in this paper. From all these experiments it can be concluded that the S-values of the various components are reproducible with a standard deviation of about 10% or less but that the number of components can differ from one preparation to another. A component with a sedimentation coefficient of about 250 S, though absent in Figure 2, can often be detected in microsomal membranes (cf. Figure 1). The opposite holds true for the 3600 S-component present in Figure 2; it is sometimes detected in nuclear membranes. The phenomenon was not restricted to the membranes from the rat, since plasma membranes from the calf and membranes of the endoplasmic reticulum from the chicken (experiments not shown) exhibited the same phenomenon. Whether this phenomenon is only restricted to fragmented biomembranes as were used in. our experiments and possibly to relatively small cellular components like excretion granulae, or that it also occurs in other intact biomembranes remains speculation. REFERENCES 1. 2. 3. 4.
Bangham, A. D. and Home, R. W.,J. Mol. Biol. 8, 660 (1964). Busch, H., Methods in Enzymology, Academic Press, New York, 1968, Vol. VI-B, p. 65. Hunter, M. J. and Commerford, S. L.,Biochim. Biophys. Acta 47, 580 (1961). Bloemendal, H., Bont, W. S., and Benedetti, E. L.,Biochem. J. 103,177 (1967). 85
5. 6. 7. 8.
86
Van Es, W. L. and Bont, W. S.,Anal. Biochem. 17,327 (1966). Palade, G. E.,Z Biophys. Biochem. Cytol. 1,567 (1955). Bont, W. S., Emmelot, P., and Vaz Dias, H.,Biochim. Biophys. Acta 173,389 (1969). Costoff, A. and McShaw, W. H.,J. CellBiol. 43, 564 (1969).
J. BOOM, W.S. BONT, H.P. HOFS, and M. DE VRIES Division of Biophysics, Antoni van Leeuwenhoek-Huis, The Netherlands Cancer Institute, Sarphatistraat 108, Amsterdam, The Netherlands
(Received 27 May, 1976) Abstract. Analytical centrifugation was used in order to investigate the size distribution of vesicles in various membrane preparations. Under certain conditions depending among others on the speed of rotation and the temperature, a sedimentation profile was observed that was characteristic for membraneous material. Since the membrane vesicles exhibited a discontinuous distribution of sedimentation coefficients it was concluded that membrane vesicles only occur in a few size classes.
I. INTRODUCTION Membranes from various parts of the cell are characterized by their chemical composition, enzymatic activities and buoyant density. When large membraneous structures are isolated, fragmentation of the membranes is unavoidable. The morphology of these fragmented membranes has been studied with the electron microscope. The finding that vesicles are often formed appears to be related to the fact that bilayers formed by hydrophobic lipids are the major constituents of biomembranes [ 1]. To our knowledge no systematic investigation concerning the distribution of vesicle sizes in preparations of biomembranes has been made.
II. MATERIALS AND METHODS
1. The Preparation o f Nuclear Membranes During the whole procedure a temperature of 2 - 4 ~ was maintained. The liver of a 3-monthsold male rat from the inbred strain R-Amsterdam was forced through a metal gauze with pores of 1 mm 2, and homogenized in 10 vol of a medium containing 0.10 M NaCI, 0.015 M EDTA, 0.75 M sucrose, 0.04 M Tris HCI, pH 7.5 (medium 2) using a teflon homogenizer of the PotterElvehjem type with a clearance of 0.5 mm during about 60 s with a velocity of 1400 rev min-1. The homogenate was centrifuged for 5 min at 2000 X g and the sediment gently resuspended in 10 vol of a medium containing 0.10 M NaC1, 0.014 M EDTA, 0.04 M Tris HC1, pH 7.5 (medium 1) (cf. [2]) using the same homogenizer driven by hand. After 20 min stirring in the cold room we centrifuged for 5 min at 2000 • g; this step was repeated once. Although the sediment was 81 Molecular Biology Reports 3 (1976) 81-86. All Rights Reserved. Copyright 9 1976 by D. Reidel Publishing Company, Dordrecht-Holland.
not free from cytoplasmic contaminants as could be seen by phasecontrast microscopy, this crude preparation of nuclei was routinely used for the extraction of membranes. The wall of the centrifuge tube was rinsed and the sediment washed with a small volume of bidistilled water. After centrifugation for 5 min at 2000 X g the sediment was carefully resuspended in 25 ml bidistilled water per liver, with the aid of a hand-driven teflon homogenizer having a wide gap between pestle and mortar (clearance approx. 1 mm). This suspension was introduced in a pressure homogenizer (Baskerville and Lindsay Ltd., Manchester) as described by Hanter and Commerford [3]. Nitrogen was slowly admitted at a rate such that it took about 5 min to reach a pressure of 800 lb inch -2. After 15 min the suspension was expelled from the pressure vessel and centrifuged for 15 min at 10 000 X g. The sediment was discarded and the supernatant was layered on 10 ml of a 2 M sucrose solution in a tube, fitted for the type 30 rotor of the Spinco Model L preparative ultracentrifuge. The tube was equilibrated with bidistilled water, and centrifuged for 30 min at 100 000 • g. The material in the interphase was collected and used for further investigations.
2. Preparation of Membranes of the Endoplasmic Reticulum Both smooth and rough membranes of the endoplasmic reticulum were isolated from rat liver cells as described previously [4].
3. Analytical Centrifugation The preparations were analyzed at 260 nm in a Spinco Model E analytical ultracentrifuge equipped with UV absorption optics and an automatic scanning system developed in this laboratory [5]. The concentrations varied from about 0.5 mg m1-1 for membranes from the endoplasmic reticulum to 0.1 mg ml-1 for plasma membranes for analysis in the ultracentrifuge. The absorbance of all preparations was between 1 and 2 units.
III. RESULTS
1. Sedimentation Analysis of Nuclear Membranes The sedimentation velocity was studied in a Spinco Model E analytical ultracentrifuge equipped with U.V. absorption optics coupled with an automatic scanning system as described previously [5]. The results of a typical sedimentation experiment are given in Figure 1. It is evident that the membrane contained well defined fractions as revealed by the steplike sedimentation pattern. The sedimentation coefficients of fractions i-7 (see Figure 1) were 131 S, 251 S, 425 S, 610 S, 869 S, 1169 S and 2163 S, respectively.
2. Sedimentation Analysis of Cytoplasmic Membranes We were interested to know whether these findings were restricted to membranes isolated from a nuclear fraction or that it was an aspect of biomembranes in general. One could argue that nuclear membranes contain DNA, be it only as a small fraction of the total mass of the membraneous material. This non-membraneous nuclear material could possibly be responsible for the peculiar fragmentation of the nuclear membranes. Therefore we decided to investigate the membranes of the endoplasmic reticulum. It is well known that the membrane fraction of 82
Fig. 1. Sedimentation analysis of nuclear membranes. The membranes were isolated as described under 'Materials and Methods' and diluted with bidistilled water to an absorbance at 260 nm of about 1.5. The sedimentation prof'des were traced at 1.5 min, 4.5 min, 13.5 min and 19.5 min, respectively, after reaching maximum speed (= 16 200 rev min-l). Temperature = 20 ~ M = meniscus at a radial distance of 5.88 cm. For the sedimentation coefficients of the 7 components see text.
the endoplasmic reticulum consists o f two groups o f membranes viz the rough membranes, i.e. membranes dotted with ribosomes, and smooth membranes, i.e. membranes without ribosomes [6]. Both smooth membranes and rough membranes were analyzed and in Figure 2 a typical sedimentation profile obtained for smooth membranes is given. The 6 components had S-values o f 150 S, 425 S, 778 S, 1016 S, 1539 S and 3653 S, respectively.
IV. DISCUSSION Membrane preparations that exist in the form of vesicles were examined in the analytical ultracentrifuge equipped with absorption optics. When analyzing the tracings, we have to keep in mind that the absorbance o f membraneous material at 260 # m was due to light-scattering, since the absorbance diminished after the addition o f a detergent, e.g. SDS, to the suspension. By the addition o f detergents membrane-vesicles are broken down into particles too small to give 83
Fig. 2. Sedimentation analysis of membranes of the endoplasmic reticulum. The membranes were isolated as described under 'Materials and Methods' and diluted to absorbance at 260 nm of about 1.5, with a medium containing 0.07 M KC1, 0.05 M Tris pH 7.6 and 8 mM MgCI.The sedimentation profiles were traced at 0 min, 3 rain and 7.5 min, respectively, after reaching maximum speed (-- 13 600 rev min-1). Temperature = 20 ~C. M = radial distance of meniscus. R = reference point at 7.3 cm. For the sedimentation coefficients of the 6 components see text. light-scattering at 260 nm [7]. Since the rough membranes o f the endoplasmic reticulum were heavily contaminated with ribosomes, part of the absorbance at 260 nm must be ascribed to the ribosomes and this in turn explains why the concentration of material corresponding to about 1 absorption unit in these preparations was higher (0.5 mg ml-1) than in the membrane preparations derived from the nucleus and the plasma membrane (0.1 mg ml-1). When complex structures like biomembranes are fragmented, one would expect a random distribution o f sizes with a mean size dependent on the mode o f preparation o f the membraneous material. With very subtle isolation methods the mean size should correspond to very large particles; a crude isolation procedure should result in relatively small particles. The fact that fragments of nuclear membranes only exist in a few classes, that are characterized by well 84
defined sedimentation coefficients, poses the question whether this is a more general phenomenon of biomembranes. In order to answer this question the vesicles of membranes were separated on sucrose gradients. The heavier fractions contained only components with high S-values, the light fractions only low S-values, and the intermediate fractions almost exclusively the intermediate S-values. In addition the S-values could be correlated with the results obtained with the electronmicroscope: the higher the S-value the larger the particles. It was therefore concluded that the sedimentation profde was not due to sedimentation artifacts. Each ~sedimenting component consists of a vesicle with definite mass. The more rapidly sedimenting components are not aggregates of smaller ones since electronmicroscopic studies revealed that the heavier components did not contain aggregates of small vesicles. Now one could argue that biomembranes have a mosaic structure and when the membranes are disintegrated, rupture leaves certain fragments of the mosaic intact. A component with a definite sedimentation coefficient should then correspond to a certain part of the mosaic. However, artificial membranes (liposomes) also exhibited a discontinuous size distribution and this again favours the assumption that the different vesicles do not represent various identical structures of a mosaic present in biomembranes. It was concluded therefore that vesicles of biomembranes occur in only a few size classes. The finding that certain cellular components, as e.g. excretion granulae, are homogeneous in size [8] might indicate that also in the intact biomembranes a purely physical phenomenon is operative for the determination of particle size. When the S-values for the microsomal membranes and the nuclear membranes were compared it was striking that certain components in the nuclear membranes had the same S-value as those in the microsomal membranes. Thus components 1, 2, 3 and 4 of the microsomal membranes had approximately the same S-value (less than 15% difference) as components 1, 3, 5 and 6, respectively, of the nuclear membranes. Only two of the many experiments (about 20 for each type of preparation) were shown in this paper. From all these experiments it can be concluded that the S-values of the various components are reproducible with a standard deviation of about 10% or less but that the number of components can differ from one preparation to another. A component with a sedimentation coefficient of about 250 S, though absent in Figure 2, can often be detected in microsomal membranes (cf. Figure 1). The opposite holds true for the 3600 S-component present in Figure 2; it is sometimes detected in nuclear membranes. The phenomenon was not restricted to the membranes from the rat, since plasma membranes from the calf and membranes of the endoplasmic reticulum from the chicken (experiments not shown) exhibited the same phenomenon. Whether this phenomenon is only restricted to fragmented biomembranes as were used in. our experiments and possibly to relatively small cellular components like excretion granulae, or that it also occurs in other intact biomembranes remains speculation. REFERENCES 1. 2. 3. 4.
Bangham, A. D. and Home, R. W.,J. Mol. Biol. 8, 660 (1964). Busch, H., Methods in Enzymology, Academic Press, New York, 1968, Vol. VI-B, p. 65. Hunter, M. J. and Commerford, S. L.,Biochim. Biophys. Acta 47, 580 (1961). Bloemendal, H., Bont, W. S., and Benedetti, E. L.,Biochem. J. 103,177 (1967). 85
5. 6. 7. 8.
86
Van Es, W. L. and Bont, W. S.,Anal. Biochem. 17,327 (1966). Palade, G. E.,Z Biophys. Biochem. Cytol. 1,567 (1955). Bont, W. S., Emmelot, P., and Vaz Dias, H.,Biochim. Biophys. Acta 173,389 (1969). Costoff, A. and McShaw, W. H.,J. CellBiol. 43, 564 (1969).
