Two Types of Vesicles

Transcription

1 Eur. J. Biochem. 41,37-43 (1974) Two Types of Vesicles from the Erythrocyte-Ghost Membrane Differing in Surface Charge Separation and Characterization by Preparative Free-Flow Electrophoresis Hans-G. HEIDRICH and Gerhard LEvrNER Abteilung Hannig, Max-Planck-Institut fur Biochemie, Martinsried bei Miinchen (Received June 21/September 5, 1973) Vesicles of erythrocyte ghosts were produced by treatment with Primaquine or hexokinase. The resulting vesicles could be separated from each other and characterized by preparative free-flow electrophoresis. One population consisted of outside-out vesicles carrying the same sialic acid distribution as the original ghosts. The other represented homogeneous inside-out particles with a different electrophoretic mobility carrying most of their sialic acid at their inner side. The two kinds of vesicles could not be separated by density gradient centrifugation. The goal of understanding the function of biological membranes can be approached by studying their chemical structure, particularly the chemical topology of both of their surfaces. Basic requirements for a comparison between these two membrane surfaces are the ability to reverse the biological membrane and, more important, the possibility of separating into homogeneous populations both the outside-out and inside-out membrane vesicles. There are published results showing that classical separation methods such as density-gradient centrifugation have been used for the separation of vesicles of the same type membrane [1,2]. However, a more direct and understandable separation method for this purpose should be based on the difference in electrical charge of the two surfaces [3]. The membrane of erythrocytes contains most of the negatively charged groups on the outer side, therefore opening up the possibility for an electrophoretic separation of the outside-out and inside-out types of membrane vesicles. In the present paper results are reported on the separation, using preparative continuous free-flow electrophoresis [4], of the two types of erythrocyte membrane vesicles. One type contains most of the sialic acid on the outer surface while the other predominantly contains the sialic acid at the inner side. These two types of vesicles could not be separated from each other by density-gradient centrifugation. EXPERIMENTAL PROCEDURE Preparation of Erythrocyte Ghosts The cells of 100 ml freshly drawn human blood were spun down at 120 xg for 10 min and then washed three times with a 5 mm phosphate buffer, ph 8.0, containing 0.15 M NaCl. The cells were hemolyzed with 1200 ml 5 mm phosphate buffer, ph 8.0, by stirring at 0 "C for 20 min. The resulting ghosts were then washed 5-6 times at 4 "C with the same buffer by centrifugation at xg for 10 min until they were white. If only ghosts without further treatment were prepared, the hemolysis and the washes were followed by a 60-min incubation at 36 "C. Treatment with Primaquine I0 ml packed ghosts in 10 ml 5 mm phosphate buffer, ph 7.4, were carefully mixed with 20 ml of a 1 mm Primaquine solution in the same buffer (Bayer, Leverkusen) and incubated at 36 C for 90 min. The ghosts were then centrifuged at x g for I0 min. The pellet was suspended in 3ml phosphate buffer and taken into a syringe through a 27-gauge hypodermic needle and slowly injected into 5 ml I0 mm triethanolamine-acetate buffer, ph 7.4, containing 0.1 mm MgSO,, and centrifuged at x g for 10 min. The pellet was suspended in 2-3 ml triethanolamine buffer con- Eur. J. Biochern. 41 (1974)

2 38 Surface Charge of Erythrocyte-Membrane Vesicles taining 0.1 mm MgSO,, and used for densitygradient runs or electrophoresis experiments. Treatment with ITP, Glucose and Hexokinase A mixture of 3.4 mg ITP (inosine 5'-triphosphate), 2 mg D( + )-glucose, 0.25 mg hexokinase, and 1 mg ghost protein in 1 ml triethanolamine buffer, ph 7.4, was incubated at 37 "C for 30 rnin and centrifuged at x g for 15 min. The pellet was then resuspended in 2 ml triethanolamineacetate buffer and homogenized with a hypodermic needle as described above. After two washes in triethanolamine-acetate buffer containing 0.1 mm MgSO,, the suspension was used for density-gradient and electrophoresis experiments. Protein Determination Protein concentration was determined with an automated Technicon AutoAnalyser as described in [5]. Sialic-Acid Determination Sialic acid was determined according to Warren [6], using 0.2 ml periodate solution instead of 0.1 ml. Density Gradient Linear gradients were prepared from 5.5 ml dextran T 110 solution (8.Og in 50ml 1OmM triethanolamine-acetate buffer, ph 7.4, containing 0.1 mm MgSO,) and 5.5 ml dextran T 110 solution (1.62 g in 50 ml of the same buffer). 1-ml sample was applied to each of the gradients and the gradient was centrifuged in an IEC B-60 ultracentrifuge at x g for 16 h. Free-Flow Electrophoresis Free-flow electrophoresis experiments were carried out in a free-flow electrophoresis IV apparatus according to Hannig [4]. The electrode vessel buffer consisted of 100 mm triethanolamine, 100 mm acetic acid, ph 7.4 (2 N NaOH), the buffer in the separation chamber was made of 10 mm triethanolamine, 10 mm acetic acid, 0.1 mm MgSO,, ph 7.4 (2 N NaOH). The conductivity of the buffer was 0.67 mmho. The conditions for the runs were as follows: 100 loo/, V/cm, 100 ma, temperature 5 "C, electrophoresis buffer flow 2.25 ml/h per fraction, sample injection above fraction 70 was 3.8 ml/h. 92 fractions were collected at 0 "C. Microscopy For light microscopy a Zeiss Photomicroscope was used with a Neofluar (phase-contrast) and a flash light. For electron microscopy the samples from the electrophoresis runs were collected in 1 ml glutaraldehyde solution, then centrifuged at x g for 30 rnin and the pellets fixed with glutaraldehyde for another hour. After fixation with OsO,, dehydration and embedding in Epon 812, thin sections were stained with uranyl acetate and lead citrate. A JEM T7 electron microscope was used for viewing the specimens (60 kv). Cleavage with Trypsin The erythrocyte ghosts and vesicles in triethanolamine-acetate buffer were incubated with trypsin at 37 "C for 60 rnin (1.25 mg Trypure Novo, Novo Industrie, Mainz, per 10 mg vesicle protein). The mixture was then centrifuged at x g for 15 min and the sialic acid was determined in the pellets and supernatants. RESULTS Morphological Evidence for Different Vesiculations It has been shown in experiments from other authors tha,t membranes of erythrocytes are able to form vesicles either by budding to the inside or to the outside of the cell followed by a re-vesiculation of the membrane. Primaquine [7] or vitamine A [8] induce budding into the cell, whereas a combination of ITP, glucose and hexokinase [9] induce vesicles to be produced on the outside. In the present study, Primaquine was used in order to obtain a vesiculation into the cell. After only 10-min incubation of the ghosts with Primaquinc, it could be observed in the phase-contrast microscope that the membranes were forming protuberances which, within about 90 rnin became vesicles inside the ghosts. Electronmicrographs from those altered ghosts illustrated small vesicles within the boundary membranes. No buds or vesicles could be observed at the outer side of the membranes (Fig. 1). In contrast, a combination of ITP, glucose and hexokinase resulted in a vesiculation to the outaside of the membrane. This could also be observed in the light microscope. Sections observed in the electron microscope demonstrated that by 30 min the boundary membrane was forming buds and vesicles only to the outside. No vesicles could be detected within the ghosts (Fig.2). After a very careful homogenization with a hypodermic needle in combination with the stabilizing addition of Mg2+, both preparations produced almost the same heterogeneous mixture of large and small vesicles. Separation in the Density Gradient Centrifugation of Primaquine-treated ghosts in a dextran gradient produced three distinct bands of vesicles (Fig.3). In the top band (band 3) large and

3 H.-G. Heidrich and G. Leutner 39 Fig. 1. Electronmicrograph of a typical, Primaquine-treated ghost. The membrane has budded to the inside and vesicles have been formed inside of the ghost. Magnification The bar represents 1 Fm Fig. 2. Electronmicrograph of a typical ITP-hexokinase-glucose-treated erythrocyte ghost. The membrane has budded to the outside and vesicles have been formed out of the ghost. Magnification The bar represents 1 pm Eur. J. Bioohem. 41 (1974)

4 40 Surface Charge of Erythrocyte-Membrane Vesicles Fig. 3. Diagram of the dextran gradient with a Primaquine-treated erythrocyte-ghost homogenate. 3 bands have been formed. The top band (band 3) and the bottom band (band 1) were subjected to electrophoresis. (-) Protein concentration of Primaquine-treated ghosts; (- --) protein concentration of untreated ghosts; (----) dextran concentration in gradient - E T 0.: 1000 c 0, n small vesicles could be recognized in phase contrast. The middle band (band 2) has not yet been characterized, while large particles and destroyed membrane material were found in the bottom band (band 1). In all experiments in which the ghosts were treated to produce vesicles either on the outside or the inner side of the ghosts, the upper band in the gradient was observed. Gradient centrifugation of untreated ghosts also produced both the top and the bottom bands. Separation in the Free Flow Electrophoresis The separation diagrams of typical free- flow electrophoresis runs are shown in Fig.4. The original ghosts are deflected from the injection port (above fraction 70) into fractions with the peak at fraction 27 (Fig.4A). The same major peak could be observed when the homogenate of the Primaquine-treated ghosts was injected into the electrophoresis but an additional peak was obtained in fractions (Fig.4R). This was shown in electronmicrographs to contain small vesicles (Fig. 5). This additional peak was almost absent when the Fraction No. Fig. 4. Separation diagrams of free flow electrophoresis runs. (A) Original erythrocyte ghosts, before or after gradient, and top or bottom band of the gradient. (B) The homogenate of Primaquine-treated ghosts. The material in the left band has the same surface charge as ghosts (outside-out), the right band vesicles have another charge and are inside-out. (C) The homogenate of Primaquine-treated ghosts after density-gradient centrifugation. (----) Top band from the gradient; (-) bottom band from the gradient

5 H.-G. Heidrich and G. Leutner 41 Fig, 5. Electronmicrograph of the inside-out vesicles from Primaquine-treated erythrocyte ghosts. (Fig. 4B and 4C right peak.) Magnification The bar represents 1 pm material in either the top of the bottom band of the dextran gradient of the untreated ghosts were subjected to electrophoresis. It was, however, clearly present when the upper gradient band of the Primaquine-treated ghosts (containing large and small vesicles) was electrophoresed ; but there was also a peak in the same region as the original ghosts (Fig.4C). The lower gradient band of the Primayuine-treated ghosts produced in the electrophoresis mostly material in the region of the untreated ghosts and with only a shoulder between fraction (Fig.4C). As was expected, both gradient bands from the hexokinase-treated ghosts had the same electrophoretic mobility as the ghosts (Fig. 4 D). Sialic Acid Content of the Surfaces of the Fractionated Vesicles The sialic acid content of the surfaces of the vesicles and the total sialic acid content after hydrolysis gave some information about the nature of the membraneous material. Note: all the particles that were isolated either by density gradient or by electrophoresis had about the same total content of sialic acid per mg protein, indicating that the same membrane systems were present. This was also confirmed by acrylaniide electrophoretic analysis of the membrane proteins not described in this paper. On their outer surface, all the membrane vesicles obtained in the electrophoresis fractions closer to the anode (24-30), had almost the same sialic acid distribution as have intact ghosts (Table 1). In contrast, all the membrane vesicles of the right band in the electrophoresis (fractions 32-34) had much less sialic acid present at the outer side of their boundary membranes although their total sialic acid content was identical with that of the total content per mg protein of the left band. DISCUSSION The budding of erythrocyte membranes either to the outer or the inner side can be controlled by chemical procedures [7-91. Theoretically, a budding to the outside results in vesicles possessing the same surface charge as have intact erythrocytes or ghosts. On the other hand, budding to the inside should result in vesicles carrying a surface charge different from intact ghosts. The only way of recognizing these facts directly is electrophoresis. In our studies, the theoretical considerations were confirmed by the experiments. The electrophoretic mobilities of both types of vesicles were different from each other but that of the outside-out population was

6 42 Surface Charge of Erythrocyte-Membrane Vesicles Table 1. The sialic acid content of trypsin-released sialopeptides from different fractions of the density gradient and the free-flow electrophoresis runs In all the experiments, the membranes after trypsin digestion were subjected to a total hydrolysis followed by a sialic acid determination in order to determine the sialopeptides at the inner membrane surface. This, plus the trypsin-released sialic acid, always resulted in approx. 10Oo/, content Treatment Density gradient Free-flow electrophoresis Sialic acid content Bands released by trypsina Vesiculation inside-out (Primaquine) top band 2 left right " bottom band 1 left 74.9 Vesiculation outside-out top band 1 left 76.2 (ITP-hexokinase) bottom band 1 left 80.9 Untreated ghosts top band 1 bottom band 1 left left Vesiculation inside-out (Prima quine ) - 2 left 77.2 right 11.1 Untreated ghosts - 1 left 87.2 a Sialic acid found in the ghosts after total hydrolysis was set as 10Oo/,. identical with that of the original ghosts. Also, the distribution of sialic acid in these particles (determined by cleaving off the sialic-acid-containing peptides from the surface with trypsin) was the same as original ghosts. In contrast, the vesicles produced by budding to the inside had many fewer sialic acid peptides on their outer side but carried them at their inner side (as was shown by total hydrolysis) and these particles had a different electrophoretic mobility from that of the original ghosts. They were deflected much less to the anode, i.e. their surface charge was much less negative, due to the sialic acid being hidden at the inner surface. A direct demonstration of these sialopeptides by a special technique for electronmicroscopy is under way. The vesicles formed were stabile in the buffers used for at least 24 h when Mg2+ was added to the preparation. From our data we cannot confirm the results of others [i j which indicate that inside-out vesicles are labile and undergo a change to outsideout particles. Also in disagreement with our findings are results of others that particles from top of the gradient are homogeneous inside-out particles [a]. In our experiments this top band, consisting of large and small vesicles, produced two distinct bands in the electrophoresis runs, one showing the charge of the original ghosts, the other that of inside-out vesicles. It is difficult to correlake directly the behaviour of the vesicles in the gradient and their behaviour in the electrophoresis. Original erythrocyte ghosts are found in both the top and bottom bands of the gradient, and have identical electrophoretic mobilities. The vesicles formed by hexokinase treatment can also be found in both bands of the gradient and all this material is deflected in the electrophoresis into the same fractions as original ghosts. In all the bands of the gradient, large and small vesicles can be found but each apparently has its own density. In order to clarify this result, the experiments of Bodemann and Passow [lo] should be discussed. According to these experiments, erythrocyte ghosts can be "re-sealed" after hemolysis by incubation to 37 "C. The re-sealed and the un-sealed ghosts can be separated from each other via an equilibrium gradient for the following reasons. The membrane of the re-sealed ghosts is tight and cannot let gradient solution pass in. Thus, those particles have a very low density and remain on top of the gradient. The leaking vesicles, however, can be continuously filled with the gradient forming solution. They obtain the high density of the lower part of the gradient and can therefore be found in the bottom band. This appears to be the reason for the presence of the two main bands in the gradient described above, and for the identical surface charge of this material with the original ghosts. In the Primaquine experiment, two bands are also produced in the gradient, the upper containing large and small intact vesicles, the lower large and small vesicles and destroyed membraneous material. In the electrophoresis, the upper band

7 H.-G. Heidrich and G. Leutner 43 is separated into two bands (Fig.4C), one having the same surface charge as have the original ghosts. This must be re-sealed ghosts or outsideout vesicles. The particles of the other peak in the electrophoresis, carry the main content of their sialic acid at the inner side. These are true re-sealed inside-out vesicles of small size (Fig.5). The lower gradient band from the Primaquine experiment (Fig.4C) is also split into two bands in the electrophoresis run : most of the material consists of un-sealed ghost or destroyed outside-out particles which are deflected into the fractions of the original ghosts. Some material which runs at the shoulder fraction must be leaking or destroyed inside-out vesicles, filled with dextran. The hexokinase treatment also results in two bands in the gradient, re-sealed and un-sealed ghosts and vesicles, but all carrying the outer side of the original ghosts to the out-side and therefore migrating in the electric field as the original ghosts (Fig.4D). As a consequence of these experiments it is apparent that gradients should not be used for obtaining homogeneous outside-out or inside-out erythrocytes vesicles. The role of Primaquine for the reaction of the erythrocyte membrane described is completely unclear but under investigation. There is definite evidence that sialic acid must be involved (a. Leutner, unpublished results). Other authors [ll] suggest that among other chemicals, lipophilic drugs with high affinities to the membrane may somehow in- fluence the stability and the pattern of the membrane, but those theories are only speculative and have to be proven. The clarification of this mechanism may be the key for a systematic reversing of biological membranes. The authors want to express their gratitude to Professor K. Hannig for his continuous interest in this work. The expert help of Mrs Illmensee and Miss Leutner during the experiments is greatly appreciated. Dr B. Olsen very kindly went over the manuscript. REFERENCES 1. Steck, T. L., Weinstein, R. S., Straus, J. H. & Wallach, D. F. H. (1970) Science (Wash. D.C.) 168, Steck, T. L., Straus, J. H. & Wallach, D. F. H. (1970) Biochim. Biophys., Acta, 203, Heidrich, H.-G. (1971) FEBS Lett. 17, Hannig, K. (1968) in Handbuch der Max-Planck-Gesellschaft, pp , Max-Planck-Gesellschaft, Munchen. 5. Heidrich, H.-G. & Hannig, K. Ado. in Automated Analysis (1970) Warren, L. (1959) J. Bid. Chem. 234, Ginn,F. L., Hochstein, P. & Trump, B. F. (1969) Science (Wash. D. C.) 164, Glauert, A. M., Daniel, M. R., Lucy, J. A. & Dingle, J. T. (1963) J. Cell Biol. 17, Penniston, J. T. & Green, D. E. (1968) Arch. Biochem. Biophgs. 128, Bodemann, H. B: Passow, H. (1972) J. Membrane Biol. 8, Wallach, D. F. H. & Weidekamm, E. (1973) Umsch. Wiss. Tech. 73, H. G. Heidrich and G. Leutner, Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Am Klopferspitz, Federal Republic of Germany