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Non-osmotic swelling in purified bovine myelin
Brain Research, 52 (1973) 97-113
97
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
NON-OSMOTIC S W E L L I N G IN P U R I F I E D BOVINE M Y E L I N
D. L. MclLWAIN* Division of Neuroscience, New York State Psychiatric Institute, New York, N.Y. 10032 (U.S.A.)
(Accepted August 31st, 1972)
SUMMARY Purified, lyophilized bovine myelin in aqueous susFension exhibits non-osmotic swelling which is influenced by the pH, ionic strength and temFerature of the bulk medium. Trypsin, chymotrypsin, papain or pronase can initiate swelling, as observed by phase contrast microscopy and turbidimetry. Swollen myelin is mechanically fragile and forms protein-deficient vesicles, some of which are not sedimented in water at 100,000 × g for 1 h. The swelling process is similar to changes reported for myelinated nerves in several experimental and pathological states and resembles two types of swelling known to occur in unilamellar membranous structures.
INTRODUCTION Swelling of the myelin sheath, accompanied by lamellar splitting and fragmentation, has been observed in demyelinating diseases 19,3a and their experimental analogues 3,27. It is not clear, however, whether these effects are directly or indirectly produced by the toxic agents used experimentally, nor are the underlying molecular changes well understood. X-ray diffraction11, a9 and electron microscopic 28 studies have demonstrated that normal myelinated nerve fibers swell in media of low ionic strength. Both techniques indicate an accumulation of water at the intraFeriod line of myelin, with splitting of the concentrically-arranged membrane pairs. Worthington and Blaurock 39 believe this type of swelling to be non-osmotic, since it can occur in hypertonic sucrose solutions and is inhibited by low concentrations of salts of divalent cations, and these authors raise the possibility that a structural change occurs within the membrane pairs during swelling. * Present address: Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
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Studies on purified membrane should permit one to investigate the direct efl'ects of swelling agents on myelin. However, a difficulty arises in the use of fresh myelin preparations for studying the swelling phenomenon. As one might expect from the electron microscopic and X-ray diffraction data cited above 11,2s,ag, myelin swells during isolation by the usual techniques in low ionic strength media. When examined by phase microscopy, myelin freshly prepared by the procedure used here shows very low refractivity, unlike the highly refractile membrane in vivo or in salt solutions 7. This change can be correlated with the swelling of myelin (see below). A recent report by De Vries et al. 7 indicates that swelling of myelin occurs within 1-2 h in ice-cold media of low ionic strength and is relatively unaffected by the osmolarity of sucrose in the medium. On the other hand, myelin which has been lyophilized and then rehydrated regains its highly refractile appearance and, more importantly, mimics a number of the features of myelin undergoing swelling and fragmentation in vivo or in isolated or cultured myelinated nerve fibers. The present study describes the swelling effects of several agents on rehydrated myelin preparations. A clearer understanding of the membrane changes described here may provide useful information not only about the nature of myelin swelling and degradation in the demyelinating diseases, but also about the molecular organization of normal myelin. MATERIALS AND METHODS
Lyophilized myelin was prepared from bovine pons and medulla by the method of Suzuki et a l ) 4 and stored at --20 °C. Lipids were extracted from myelin (12.4 mg dry weight) after first dissolving it in 1 ml chloroform-methanol (2:1) and evaporating to dryness 3 times, causing approximately 75 % of the protein to adhere to the test tube. The chloroform-methanol (2:1) soluble material was then placed on a silicic acid column (1 g) and eluted with 12 ml methanol. Quantitative yields of myelin lipid were obtained, while 92 % of the protein, as measured by the method of Lowry et al. z° was removed by this technique. Trypsin (crystallized × 2), chymotrypsin (crystallized × 3), phospholipase C (C1. perfringens) and galactose oxidase were obtained from Worthington Biochemical Corp., Freehold, N. J. ; pronase (B grade) and papain (african papaya), from Calbiochem, Los Angeles, Calif.; soybean trypsin inhibitor from Armour Laboratories, Chicago, 111.; ovomucoid (chicken) trypsin inhibitor, a-N-benzoyl-D,L-arginine-pnitroanilide HC1 and Dextran 15 (average mol. wt., 20,000), from Sigma Chemical Co., St. Louis, Mo. Diphenylcarbamylfluoride was a gift from Dr. Norbert Wassermann. Dextran 10 (number average mol. wt., 5,700) was purchased from Pharmacia Fine Chemicals, Inc., Piscataway, N.J. Phase contrast microscopy
Freeze-dried myelin suspended by gentle (bath type) sonication in water (2 mg/ml) and combined with an equal volume of the appropriate test solution was placed between two plastic coverslips, resting on a glass slide, and viewed with a Leitz Ortholux phase contrast microscope. Plastic coverslips were used to avoid pH
NON-OSMOTIC SWELLING IN MYELIN
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increases reported in aqueous films between glass surfaces 26. Coverslip effects, including increased myelin swelling, were nonetheless noted in samples which were allowed to stand for over 15 min. Thus, specimens were viewed and photographed no later than 5 min after mounting. Myelin lipids (10/~g) were dried on a glass slide and viewed under low power after introducing a drop of the appropriate buffer onto the sample and covering it with a glass coverslip. By using buffered systems and comparing the results obtained with and without a coverslip, artifacts arising from pH alterations were avoided.
Turbidimetry Optical density measurements of myelin suspensions were carried out at 520 nm, using a Beckman Model B spectrophotometer. Myelin (2 mg/ml) was suspended in deionized water by gentle sonication, and the O.D. recorded 15 min after adding an equal volume of a solution of appropriate pH and ionic strength. The pH of the sample was measured immediately after determining the O.D.
Gas-liquid chromatography ( GL C) Methyl esters of free fatty acids were prepared by treating myelin lipids (750 #g) with diazomethane in 0.5 ml cold methanol--diethylether (1:9). Palmitic acid was included as an internal standard. Methyl esters were injected onto a 15 ~ EGSS-X on 100/120 GasChrom P column (Applied Science Labs., State College, Pa.), using an Aerograph Hi-Fy apparatus (hydrogen flame ionization detector) operated isothermally at 180 °C. Peak areas were measured by multiplying the height times the width at one-half peak height. Galactose was measured quantitatively by GLC, using the procedure of Vance and Sweeley35. A 3 ~ OV-1 100/200 GasChrom Q glass column at 160 °C was employed, with mannitol as an internal standard. Quantitative thin-layer chromatography was used to separate myelin phospholipids, which were assayed for P as described by Mcllwain and Rapport 22. Cholesterol was estimated by the method of Schoenheimer and Sperry 30 and total reducing sugar by the phenol-sulfuric acid assay 17. RESULTS
Types of swelling Purified myelin, suspended by sonication in aqueous media, formed multilamellar particles 1-10 #m in diameter 22 which then aggregated to produce clumps visible by phase contrast microscopy (Fig. 1). Changing the pH of the suspension not only altered the degree of clumping, but also caused the particles to swell in many instances. Two morphologically different types of swelling were observed: (1) a roughly symmetrical form at pH < 3 or > 8 (Fig. 2) and (2) evaginative forms at pH 6-8, appearing as bulbous or elongated outgrowths from the myelin (Fig. 3). The former involved all myelin particles, while the latter did not always do so. Symmetrical swelling was accompanied by an apparent decrease in the refractivity of the fragments, giving them a dark grey appearance by phase microscopy, while the evaginations
Fig. 1. Phase contrast appearance of myelin suspended in water. Freeze-dried myelin was sonicated in deionized water (I mg/ml) and viewed between plastic coverslips. 500.
Fig. 2. Symmetrically swollen myelin. Same sample as in Fig. I, immediately after addition of 10/d of 0.1 N HC1 between coverslips. , 500.
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Fig. 3. Evaginative swelling in myelin. Freeze-dried membrane was sonicated in water (1 mg/ml) and diluted with an equal volume of 20 m M Tris-HC1 buffer (pH 8.1). Phase contrast micrographs were made of samples 30 min after preparation (25 °C). x 500.
often arose from highly refractile clusters of myelin and in the presence of salts (see below) sometimes appeared thick-walled and refractile themselves. The elongated, grey evaginations were mobile and fragile, often pinching off spontaneously to form free-floating vesicles. The long evaginations were never observed arising from symmetrically swollen particles.
Effects of pH The relationship between the pH of the medium and the phase contrast appearance of dispersed myelin in the absence of added salts can be summarized as follows: at pH < 3 the myelin particles were well dispersed and exhibited symmetrical, but not evaginative swelling. Between pH 3 and 4.5 clumping increased markedly, with no sign of swelling of the particles. Clumping decreased from pH 4.5 to 7.0 and elongated forms were noted between pH 6.0 and 8.0. The evaginations appeared within 5 rain at pH 7.4 and increased in size and number over a period of several hours. At pH 8-11, symmetrical swelling was rapid and obvious, although in the presence of salts, evaginations also occcurred above pH 8. The refractivity of the myelin particles decreased above pH 8.0, and above pH 10 many myelin particles disintegrated into very small, grey vesicles, which floated in the medium.
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pH Fig. 4. Relationship between the pH and optical density (520 rim) of aqueous myelin suspensions. Myelin was suspended in water and adjusted to the desired pH with dilute HC1 or NaOH to give membrane concentrations of 0.125 mg/ml (r-7 17); 0.25 mg/ml (O O); and 0.50 mg/ml (~ A). The O.D. is expressed relative to that for a like concentration of myelin in 8 mM imidazole-HC1 buffer at pH 7.0. Each point represents one sample. Closed circles ( 0 - - - - 0 ) denote samples of myelin (0.25 mg/ml) suspended in 2 mM CaCI2, final concentration, and adjusted to the indicated pH.
These changes were correlated with alterations in the O.D. of the suspension at 520 nm (Fig. 4). Samples containing different concentrations of myelin were adjusted to p H values from 2 to 11. A decrease in O.D. would be expected either with an increase in clumping of the particles or with swelling of the myelin. Decreases in O.D. based on particle collisions (clumping) should vary as a function of the myelin concentration, unlike swelling 4°. As can be seen (Fig. 4), differences in the percent decreases in O.D. of the 3 suspensions occurred only between p H 3 and 7, the range in which clumping was observed microscopically. Below p H 3 and above p H 7, however, these differences disappeared, indicating that decreases in O.D. in these ranges reflected swelling of the myelin particles. The two types of swelling could not be distinguished by the O.D. changes shown in Fig. 4. However, the rapid decrease in O.D. above p H 9.5 appeared to correlate with the onset of disintegration of the symmetrically swollen myelin. The decrease in turbidity of myelin suspension at pH 9 was partially reversed by back titration with dilute HC1, while at p H 2-3 the O.D. did not increase with N a O H addition. Myelin preparations which did not disperse well in water gave similar, but somewhat flatter curves than shown in Fig. 4. Less than 1.6/zg of free fatty acids and aldehydes per mg dry myelin were detected by G L C of lipid extracts from suspensions previously adjusted to p H 1.9 with HCI
NON-OSMOTIC SWELLING IN MYELIN
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or to pH 10.5 with NaOH (and in control samples at pH 7.1) and then extracted with 4 vol. of chloroform-methanol (2 :I) at pH 4.0. If liberated during this procedure, these long-chain products would account for the cleavage of less than 2 % of acid and alkali labile phosphatides in myelin. Moreover, no changes in phosphatide composition were found by quantitative TLC of myelin exposed to the same extremes of pH and compared to control samples at pH 7.1.
Salt effects The presence of salts affected aqueous suspensions of myelin in several ways. First, the salts of monovalent and divalent cations promoted clumping of the particles. This effect was clearly seen with 8 mM imidazole-HC1 buffers (pH 7.0) containing 100 mM NaC1, 100 mM KCI, 5 mM CaCI2 or 5 mM MgCI2. Secondly, these salts retarded swelling at alkaline pH. For example, inhibition of swelling was observed with 5 mM CaC12 or MgC12 and with 1 M NaCI or KC1 between pH 7.0 and pH 9.0. NaC1 and KCI (100 mM) were much less effective in this regard than 5 mM CaC12 or MgC12 at pH 9.0. Thirdly, in certain instances salts influenced the morphology of swollen myelin. In 0.5 mM CaC12 at pH 8.0 highly refractile evaginations were often produced, unlike the grey ones seen in the absence of CaC12. Moreover, evaginative, rather than symmetrical swelling was observed between pH 8 and 11 when 5 mM CaCI2 was present. The O.D. changes in myelin suspensions containing 2 mM CaC12 and adjusted to various pH values are shown in Fig. 4. It can be seen that 2 mM CaCI~ retarded swelling between pH 7 and 11 much more than at low pH and promoted clumping between pH 3 and 7.
Time and temperature effects The temperature of the myelin suspension during incubation also affected the degree of swelling visible by phase microscopy. When myelin (1 mg/ml) was suspended in 8 mM imidazole-HC1 (pH 7.4) at 5 °C and aliquots were taken for subsequent incubation at 5 °C, 37 °C and 90 °C for 10 rain, increases in evaginative swelling varied directly with the temperature: while little apparent swelling took place at 5 °C, many long, thin evaginations were observed at 90 °C, with a moderate number appearing at 37 °C. In addition, swelling slowly increased with time in these samples. As expected, the O.D. of such suspensions decreased with increasing time and temperature, but this was found (by varying the myelin concentration) to be caused, in part, by increased clumping of myelin. Aqueous suspensions of myelin at 5 °C showed decreases in O.D. equal to those seen at 25 °C (Fig. 4) as their pH was increased from 7 to 9. That swelling occurred at such extremes of temperature and pH militated against an enzymatic basis for the process.
Swelling of myelin lipids The morphology of evaginative forms of swelling already described was strikingly similar to 'myelin forms' known to arise from a variety of lipid emulsions. In order to compare the swelling of myelin lipids with that of the parent membrane, myelin
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lipids were exposed to media of varying pH and ionic strength. In deionized water, tubular formations appeared almost instantly and grew exuberantly under the coverslip for several minutes. The forms resembled those of myelin membrane suspensions in several ways: they presented a large variety of elongated and bulbous shapes, had the same grey hue of the membrane evaginations, were highly mobile and capable of spontaneously vesiculating. Furthermore, their appearance was retarded by the presence of salts in the medium. Solutions containing l0 mM CaCI2 or MgCI2 markedly inhibited the development of such forms, while as little as 1 mM CaCl2 substantially reduced the swelling. NaCI and KC1 (0.5-1 M) showed inhibitory effects comparable to those to 5 mM CaCI2. Moreover, these salts altered the morphology of the orms, making them shorter, thinner and more refractile, as they did evaginations from the membranes. Besides their more rapid swelling, myelin li~'ids difJered from the parent membranes in their responses of pH changes. In media of low ionic strength myelin lipids exhibited swelling which did not vary noticeably between pH 3 and 11. Two different aqueous s y s t e m s - citric acid-Na2HPO4 buffers (10-15 mM); and glycine-HCl, NaOAc, and imidazole-HC1 buffers (10 raM) --were employed in these experiments. In contrast, 20 mM HC1 (pH 1.8) retarded swelling of the liFids, lzroducing highly refractile, slowly develoring forms.
F~{/bct of proteases The differences between myelin lipids and membranes with respect to pH effects on swelling suggested that myelin proteins played a role in the phenomenon. Much stronger evidence for this p~ssibility was obtained in experiments with several proteolytic enzymes. Myelin (1 rag) suspended in I ml of 8 mM imidazole-HCl buffer at pH 7.0, containing 20 #g of trypsin/ml, showed marked evaginative swelling within 10 rain at 25 °C (Fig. 5), whereas samples containing no enzyme or enzyme preincubated with one molar equivalent of soybean trypsin inhibitor were only slightly swollen. Trypsin-induced swelling was inhibited by 5 mM CaCI2, but not by 2 mM Dextran 10. As little as 1 #g of trypsin/ml produced evaginations under these conditions. A much greater decrease in O.D. occurred in myelin suspensions exposed to trypsin than in samples with soybean trypsin inhibitor (STI) or no enzyme (Fig. 6). One to 11 molar equivalents of diphenylcarbamylfluoride(DPCF) 9, preincubated with trypsin for 30 min at 25 °C, caused 80-90% inhibition of the initial rate of myelin swelling and inhibited the hydrolysis by trypsin of fl-N-benzoyl-D,L-arginine-pnitroanilide-HCl1° to a comparable extent. On the other hand, 1-10 molar equivalents of ovomucoid trypsin inhibitor (OTI) reduced the initial rate of myelin swelling 70-80 %, while equimolar quantities of the inhibitor caused 98 % inhibition of trypsin hydrolysis of the aforementioned synthetic substrate. No swelling or clumping resulted from exposure of myelin to OTI alone. Turbidimetric and microscopic effects very similar to those above were obtained with identical concentrations of chymotrypsin (pH 7.0), pronase (pH 7.0) and papain (pH 6.5). Heating chymotrypsin and pronase at 100 °C for 10 rain reduced the initial rate of swelling to 20 ~o or less of that seen with the untreated enzymes, as did exposure
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NON-OSMOTIC SWELLING IN MYELIN
Fig. 5. Phase contrast micrograph of trypsin-treated myelin. Membrane was suspended i n water and diluted with imidazole-HCl buffer (pH 7.0) to give 1 mg myelin per ml 8 m M buffer. The sample was then exposed to trypsin (20 #g/ml) for 10 min at 25 °C. × 500.
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Fig. 6. Trypsin-induced decrease in turbidity of myelin suspensions. Membrane was suspended in 8 m M imidazole-HC1 buffer (pH 7.0) as in previous figure. Trypsin (20/~g/ml, final concentration) was added and the O.D. changes were recorded at 1 min intervals. Trypsin plus myelin ((3 ©); trypsin preincubated with equimolar STI for 15 min at 25 °C and added to myelin suspensions (20 Fg enzyme/ml) (O Q); myelin without enzyme ([] []).
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of chymotrypsin to one molar equivalent of DPCF. The papain effect (20 big enzyme/ mg myelin/ml) was completely eliminated by 100/zM p-chloromercuribenzoic acid (PCMB). When used at the above concentrations, D P C F and PCMB did not inhibit the swelling process directly, since they showed no inhibition ofpronase-induced swelling. Phospholipase C (CI. perfringens), which can cleave 64~o of the myelin phospholipids 22, did not cause swelling under similar conditions. The question arises as to whether an osmotic mechanism could account for the swelling produced by the above agents. Osmotically active molecules previously trapped within the myelin particles could not be responsible, since their effects would have been exerted immediately upon suspension of myelin in water, before addition of the swelling agent. Donnan effects are also not likely, because swelling was induced by raising the temperature of myelin suspended in distilled water only. On the other hand, it is possible that the agents release membrane protein into the intralamellar spaces, causing water to enter, separate the membranes and increase the volume of the particles. The data on fragmentation of swollen myelin (see below) make this a plausible explanation. In order to test this, the maximum concentration of protein available for release (250 #g/mg dry myelin; predominantly 18,000-34,000 tool. wt.) was estimated by assuming that all the protein is released into the aqueous volume represented by the difference between the water space before and alter swelling. This volume difference was determined gravimetrically on pellets from suspensions of myelin at p H 6.8 and 10.8 and found to be 6.5 ~ 0.4/zl/mg dry myelin, representing a 250~,, increase in volume during swelling. A maximum concentration of approximately 1 m M protein was then calculated. Using this figure, an attempt was made to reverse swelling osmotically with a non-penetrating molecule added to the swollen myelin at comparable concentrations. Table I shows the results of such experiments. When 2 m M (final concentration) Dextran 10 (number average tool. wt. 5,700) was added to myelin swollen at pH 10.1, no reversal of swelling was noted. Similar negative results were obtained with the larger Dextran 15 (average mol. wt. 20,000). The situation was different at pH 2, however, where both dextran solutions, particularly Dextran 15,
TABLE I EFFECT OF DEXTRANS ON MYELIN SWELLING
Myelin (1 rag) was suspended in 1 ml water and diluted with an equal volume of 17 mM imidazoleHCI buffer (pH 7.0), 17 mM Na~B407 buffer (pH 10.3-10.5) or 0.02 N HCI. After mixing, 1 ml of the appropriate buffer, with or without 4 mM Dextran 10 or 15, was added. The final pH was then measured and the O.D. at 520 nm recorded. Percent decrease in O.D. is given relative to the O.D. of the suspensions at pH 7.0. After swelling, added
Buffer only Buffer + Dextran 10 Buffer ÷ Dextran 15
% Decrease in O.D. at: pH 2.0
pH 10.1
40.4 ± 5.8 (S.D.) 33.4 ± 4.0 14.6 ± 4,7
29.6 4- 2.3 35.9 ± 2.2 33.5 ± 5.2
NON-OSMOTIC SWELLING IN MYELIN
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Fig. 7. Electron microscopic appearance of membranous fragments which did not sediment in water at 56,500 x g for 15 min. The sample was prepared by a two-step centrifugation technique from myelin swollen at 38 °C for 1 h in 6 mM imidazole-HCl buffer (pH 7.4) containing 2.2 mM CaCI2. After sedimentation at 56,500 x g for 15 min, the pellet was suspended in water by sonication and recentrifuged at 56,500 x g for 15 rain. The vesicles, found in the second supernatant fraction, were sedimented in 20 mM CaCI2 and the pellet was then fixed in 3 ~ glutaraldehyde (pH 7.4) and stained with uranyl acetate-lead citrate, x 26,300. caused a reversal o f the relative O.D. Decreasing the p H o f the d e x t r a n - c o n t a i n i n g solutions to p H 1.7 d i m i n i s h e d the O . D . and p r o d u c e d symmetrically swollen particles similar to those p r e s e n t before a d d i t i o n o f the polysaccharide. Thus, swelling at acid p H c o u l d p r o c e e d in the presence o f the Dextran, b u t r e q u i r e d a lower p H t h a n in the absence o f the D e x t r a n . The reason for this change is n o t clear. In a n y case, the results indicate t h a t swelling o f myelin at b o t h p H extremes is largely a n o n - o s m o t i c
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process. The lack of effect of 2 mM Dextran 10 on trypsin-induced myelin swelling (see above) further supports such an interpretation.
Fragmentation of swollen myelin Myelin exhibiting evaginative or symmetrical swelling could vesiculate spontaneously. Phase contrast microscopy showed that sonication of these swollen preparations markedly increased vesiculation and reduced the size of the fragments formed. The vesicles, which could be produced from myelin swollen under quite mild conditions, were separated from the remaining myelin particles by centrifuging the sonicated preparation in water. For example, myelin showed moderate swelling when incubated at 38 °C for 1 h in 6 mM imidazole-HCl buffer (pH 7.4) containing 2.2 m M CaCl2 (ef. ref. 22, Fig. 2). The sample was then sedimented at 56,500 × g for 15 rain and the supernatant fraction removed. Upon sonication of the pellet in water and recentrifugation at 56,500 × g for 15 rain, vesicles approximately 0. l-0.5/zm in diameter (Fig. 7) were separated from the large particles. The vesicles, which were precipitated with 10 m M CaC12 before electron microscopy, appeared to exist, as such, before addition of the salt: 70 ~o were removed from water by passing the suspension through a cellulose acetate (Millipore) filter with an average pore diameter of 0.22/~m, while 39 ~o would not pass a filter with an average pore diameter of 0.45 /~m. When the filters were soaked in such suspensions for 1 h, 11 ~o of the fragments adhered to them, indicating some non-specific binding. The relationship between swelling and vesiculation was studied by the two-step centrifugation technique employed above, which allowed all samples of swollen myelin to be sonicated and centrifuged in the same medium (water). The quantity of non-sedimented phospholipid in supernatant 2 (Table II) was directly related to the p H at the time of swelling, when studied between pH 6.8 and 11.2. The percent total
TABLE II ANALYSIS OF NON-SEDIMENTABLE MYELIN PHOSPHOLIPID AND PROTEIN
Myelin suspensions at designated pH were centrifuged at 56,500 × g for 15 min (5 °C). Pellets were resuspended by sonication in water and centrifuged a second time at 56,500 x g for 15 min. In sample 7, myelin suspensions in 8 mM imidazole-HC1 (1 mg/ml) were first treated with trypsin (20 ttg/ml) at 25 °C for 30 rain.
Sample No.
1 2 3 4 5 6 7
deionized water, aqueous NaOH, aqueous NaOH, aqueous NaOH, aqueous NaOH, aqueous NaOH, trypsin (20 #g/ml),
Supernatant 1
Supernatant 2
°/o total phospholipid
Protein/P % total Protein/P (w/w) phospholipid (w/w)
pH 6.8 12.4 ± 0 pH 7.1 15.0 ~ 0.7 pH 7.9 14.8 ± 0.6 pH 9.6 13.2 ~- 1.0 pH 10.5 18.8 ± 4.5 pH 11.2 14.9 ~ 0.3 pH 7.0 13.0 ± 1.2
3.5 5.0 8.5 10.6 14.4 25.0 --
6.2 ± 8.7 ± 15.0 i 25.2 ~ 28.2 ± 33.6 ± 31.0 ±
1.3 0.6 0.01 1.1 3.0 3.9 2.9
11.2 3.8 8.1 7.5 10.7 13.3 6.7
NON-OSMOTIC SWELLING IN MYELIN
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phospholipid in the first supernatant was approximately the same for each sample and probably represented fragments originally present in the myelin preparations. Even when centrifugation was performed at I00,000 × g for 1 h, 10-15~ of the total phospholipid still remained in supernatant 2, after swelling at pH 10.7 or under conditions used to produce the fragments shown in Fig. 7. The protein :phosphorus ratio in freeze-dried myelin was 16:1. It can be seen (Table II) that all the supernatant fractions, particularly those samples between pH 7 and 10, were deficient in protein, except sample 6, supernatant 1. Vesicles of similar composition were isolated from myelin swollen by treatment with trypsin (sample 7, Table II). The protein:P ratio in supernatant 1 increased as the pH was raised, possibly indicating solubilization of protein at higher pH values. A similar pattern was observed in supernatant 2, except between pH 6.8 and 7.1. The initial protein content was completely accounted for in the supernatant and pellet fractions. Phospholipid: galactolipid : cholesterol ratios in the vesicles and in pellet fractions from samples treated as that in Fig. 7 or swollen at pH 10.7 were similar to the ratios found in lyophilized myelin (1.9:1.1:1.0). DISCUSSION
Evidence supporting these major points has been presented: (1) isolated myelin can swell by other than an osmotic mechanism; (2) two forms of swelling were noted, and (3) both were pH-dependent; (4) proteases and elevated temperature enhanced swelling, while salts were inhibitory at the concentrations studied; (5) the process could be monitored quantitatively by turbidimetry; (6) swollen myelin was mechanically fragile, forming protein-deficient vesicles which were not easily sedimented by high-speed centrifugation. The similarity in the effects of swelling agents on rehydrated myelin to those reported for intact myelin and a number of other membranes, discussed below, strongly suggests a common mechanism of swelling. Swelling in purified myelin resembled that of intact medullated fibers in being non-osmotic 89, sensitive to salts, especially those of divalent cations 3s, and to trypsin ~. The decrease in refractivity of myelin observed by Masurovsky and Bunge 21 during swelling of cyanide-poisoned, cultured peripheral nerve fibers is strikingly similar to that described here. In view of the marked alkalinizing effect of NaCN, it is possible that at the concentration of NaCN (10 mM) used in that study a pH effect was superimposed on the expected metabolic poisoning. Whether the two processes are identical must await a complete ultrastructural analysis of the changes in purified myelin and a detailed comparison of the effects on each by the swelling agents described here. Swelling and fragmentation of myelin have been reported in multiple sclerosis lesions 33. In a series of histologic investigations Hallpike, Adams and Bayliss have demonstrated an increase in proteolytic activity in demyelinating tissue in multiple sclerosis 14 and Wallerian degeneration15. They have also described the early appearance in degenerating nerve oI 'myelin buds', presumably similar to the evaginations shown here, which they attributed to increased proteolytic activity in the tissue 16.
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Moreover, fusiform swelling of myelin and subsequent fragmentation were prominent features in cultured myelinated nerve fibers exposed to antisera from rabbits with experimental allergic encephalitis a, sera from patients with multiple sclerosis is, sera containing antigalactocerebroside antibodies 8, and to several other demyelinating agents 5,~12-5. It is reasonable that the swelling process described here could be related to these pathological situations and may provide a useful way to discriminate between direct and indirect effects of these toxic substances on myelin. Similar forms of swelling are known to occur in structures bounded by a single membrane, and these are apparently initiated by changes within the membrane itself. One general class may involve a change in the conformation of membrane protein leading to alteration of membrane size and shape. Examples of this type include the Ca2+-activated contraction of erythrocyte ghosts 24 and the pH-dependent swelling of submitochondrial particles 40. Using optical rotatory dispersion and light-scattering techniques in the latter study, Wrigglesworth and Packer attributed increases in size of the unilamellar particles at high pH to disaggregation of membrane protein. A relationship between the mechanism of swelling in that system and in myelin is suggested by the similarity of the light-scattering-pH profile of submitochondrial particles to the O.D.-pH profile of myelin (Fig. 4). That changes in myelin protein were implicated in the swelling process may also be inferred from the differences in swelling of myelin lipids and myelin membranes - - the former occurring more rapidly and largely independent of pH - - and, more directly, from the fact that proteolytic enzymes also produced evaginations. Incomplete inhibition of trypsin-induced swelling by OT1 and DPCF left open the possibility that hydrolysis of protein was not required for the effect. However, it is noteworthy that myelin basic and acidic proteins, but not proteolipid protein, are susceptible to cleavage by trypsin la. Other factors (temperature, salts) shown here to influence swelling also may have exerted their effects, at least in part, on myelin protein. The protein possibly inhibited the swelling process in some way, and an alteration in myelin protein may have been an early event in the changes noted. Another similar type of swelling in biological membranes resembles the wellknown 'myelin forms' arising from lipid emulsions. Evaginations, termed 'stromatolytic forms', have been produced from erythrocyte ghosts under conditions where swelling occurred against or without an apparent osmotic gradient 12. Furchgott suggested that the molecular changes occurring within the ghost membrane involved a reorganization of lipid and possibly protein lz. Similar forms have been observed arising from a number of tissues and cells, including myelinated nerve fibers 12,a6, grey and white matter 6,al, liver slices 2a, erythrocytes ~,2, and leukocyteszg. In the present study the evaginations were like 'myelin forms' produced from suspensions of myelin lipids both morphologically and in their response to salts. The relationship of evaginative to symmetrical swelling and their comparative effects on the turbidity of the suspensions remain undefined, although the two types of swelling may represent different phases of the same process, with evaginations resulting earlier from focal changes in the membrane. What then can be said with regard to the mechanism of swelling in myelin? It
NON-OSMOTIC SWELLINGIN MYELIN
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seems probable that changes in the organization of both myelin protein and lipid accompany swelling, with alterations in protein possibly initiating the process. Moreover, the marked decrease in protein: P ratios in membrane fragments which were swollen at high pH or by exposure to trypsin indicates that the molecular disruption was extensive. However, in only one instance (protease treatment) was there reason to suspect cleavage of covalent bonds during swelling. The question then arises as to whether these changes represent a rearrangement of the myelin layers (e.g. an unrolling or fusion of the lamellae) or an actual increase in the surface area of the membrane. In the case of the unilamellar submitochondrial particles 4° surface area expansion seemed to occur at pH 9. This suggests a like mechanism for myelin. However, the answer must await more direct experimental support. Since the O.D. of myelin suspensions varied as a result of effects of pH and ionic strength on components of the membrane, there is reason for caution in the use of tubidimetry (or other light-scattering methods) for measuring osmotically-induced volume changes in membranous structures. A purely passive role for the membranes of myelin, submitochondrial particles and possibly other structures in osmotic gradients and at different pH values cannot be safely assumed. That membranes fragment in media of low ionic strength or high pH is well known 37, although the details of the process are not. In the present study fragmentation of myelin was correlated with pH- and protease-induced swelling of the membrane. It would be of interest to know if fragmentation of other membranes (e.g. red cell ghosts 4,a2) under similar conditions is also preceded by swelling. The decrease in both size and protein content of the vesicles produced from swollen myelin probably accounted for their not being sedimented under the conditions described here. Such a process could easily lead to contamination of supernatant fractions in brain homogenates, since many of the myelin fragments were not sedimented in water at 100,000 × g for 1 h. Moreover, the fact that membranous structures appeared in these high-speed supernatant fractions underlines the ambiguity of the term 'soluble' frequently applied to such fractions. It will, therefore, be of considerable importance to determine whether similar processes actually occur during preparation of myelin or other biological membranes in media of low ionic strength, such as sucrose. ACKNOWLEDGEMENTS The author thanks Dr. M. M. Rapport for his support of this study and Dr. Leon Roizin and Mr. Jevons Liu for performing the electron microscopy.
REFERENCES 1 BAKER,R. F., The fine structure of stromatolytic forms produced by osmotic hemolysis of red blood cells, J. Ultrastruct. Res., 11 (1964)494-507. 2 BESSIS,M., Phase contrast microscopy and electron microscopy applied to blood cells, Blood, 10 (1955) 272-286. 3 BORNSTEIN,M. B., AND APPEL, S. H., Tissue culture studies of demyelination, Ann. N.Y. Acad. Sci., 122 (1965) 280--286.
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4 BRAMLEY,Y. A., COLEMAN,R., AND FINEAN,J. B., Properties oferythrocyte membranes, Biochim. biophys. Acta (Amst.), 241 (1971) 752--769, 5 BUNGE, R. P., Myelin degeneration in tissue culture, Neurosei. Res. Progr. Bull., 9 (1971) 496~498. 6 CASPER,J., AND WOLMAN, M., Demonstration of myelin figures by fluorescence in some pathological tissues, Lab. Invest., 13 (1964) 27-31. 7 DE VINES, G. H., NORTON, W. T., AND RAINE, C. S., Axons: Isolation from mammalian central nervous system, Science, 175 (1972) 1370-1372. 8 DUBOIS-DALCQ,M., NIEDIECK, B., AND BUVSE, M,, Action of anti-cerebroside sera on myelinated nervous tissue cultures, Path. Europ., 5 (1970) 331-347. 9 ERLANGER, B. F., AND COHEN, W., Specific inactivation of chymotrypsin by diphenylcarbamyl chloride, J. Amer. chem. Soc., 85 (1963) 348-349. 10 ERLANGER, B. F., KOKOWSKY, N., AND COHEN, W., The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem., 95 (1961) 271-278. 11 FINEAN, J. B., AND MILLINGTON, P. F,, Effects of ionic strength of immersion medium on the structure of peripheral nerve myelin, J. biophys, biochem. Cytol., 3 (1957) 89-94. 12 FURCHGOTT,R. F., Observations on the structure of red cell ghosts, Cold Spring Harbor Syrup. Quant. Biol., 8 (1940) 224-232. 13 GONZALES-SATRE, F., The protein composition of isolated myelin, J. Neurochem., 17 (1970) 1049-1056. 14 HALLPIKE, J. F., ADAMS, C. M. W., AND BAYLISS, O. B., Proteolytic activity around multiple sclerosis plaques, Histochem. J., 2 (1970) 199-208. 15 HALLPIKE, J. F., ADAMS, C. M. W,, AND BAYLISS,O. B., Neutral and acid proteinases in early Wallerian degeneration, Histochem. J., 2 (1970) 209-218. 16 HALLPIKE,J. E., ADAMS,C. M. W., AND BAYLISS,O. B., Proteolysis of normal myelin and release of lipids by extracts of degenerating nerve, Histochem. J., 2 (1970) 315-321. 17 HAMILTON,J. K., REBERS,P. A., DUBOIS, M., GILLES, K. A., AND SMITH, F., Colorimetric method for determination of sugars and related substances, Analyt. Chem., 28 (1956) 350-356. 18 KIM, S. U., MURRAY, M. R., TOURTELLOTTE,W. W., AND PARKER,J. A., Demonstration in tissue culture of myelinotoxicity in cerebrospinal fluid and brain extracts from multiple sclerosis patients, J. Neuropath. exp. Neurol., 29 (1970) 420-431. 19 LAMPERT, P. W., Fine structural changes of myelin sheaths in the central nervous system. In G. H. BOURNE(Ed.), The Structure and Function of Nervous Tissue, Vol. I, Academic Press, New York, 1968, pp. 187-204. 20 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 21 MASUROVSKY,E. B., AND BUNGE, R. P., Patterns of myelin degeneration following the rapid death of cells in cultures of peripheral nervous tissue, J. Neuropath. exp. Neurol., 30 (1971) 311-324. 22 MCILWAIN, D. L., AND RAPPORT, M. M., The effects of phospholipase C (Cl. perfringens) on purified myelin, Biochim. biophys. Acta (Amst.), 239 (1971) 71-80. 23 PALADE,G. E.. AND CLAUDE,A., The nature of the Golgi apparatus, J. Morphol., 85 (1949) 35-69. 24 PALEK,J.,CURIIY,W.A.,AND LtONETTI,F.J.,Calcium ATPaseandcalcium-linkedghost shrinkage, Amer. J. Physiol., 220 (1971) 1028-1032. 25 PETERSON,E. R., AND MURRAY, M. R., Patterns of peripheral demyelination in vitro, Ann. N. Y. Acad. Sei., 122 (1965) 39-50. 26 PONDER, E., Hemolysis and Related Phenomena, Grune and Stratton, New York, 1948, p. 41. 27 RAINE, C. S., AND BORNSTEIN,M. B., Experimental allergic encephalomyelitis: an ultrastructural study of experimental demyelination in vitro, J. Neuropath. exp. Neurol., 29 (1970) 177-191. 28 ROBERTSON,J. D., Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions, J. biophys, biochem. Cytol., 4 (1958) 349-364. 29 ROZENSZAJN,L. A., EPSTEIN, Y., AND FISCHER, D., Myelin figures in blood cells, lysosomes and lipidoses, Amer. J. clot. Path., 52 (1969) 473-476. 30 SCHOENHEIMER,R., AND SPERRY, W. M., A micromethod for the determination of free and combined cholesterol, J. biol. Chem., 106 (1934) 745-760. 31 SHANKLIN,W. M,, Myelin figures demonstrated with the phase microscope, Stain Teehnol., 36 (1961) 377-378. 32 STECK,T. L., WEINSTEIN,R. S., STRAUSS,J. H., AND WALLACH,D. F. H., Inside-out redcell membrane vesicles: preparation and purification, Science, 168 (1971) 255-257. 33 SUZUKI,K., ANDREWS,J. M., WALTZ,J. M., AND TERRY, R. D., Ultrastructural studies of multiple sclerosis, Lab. Invest., 20 (1969) 444-454.
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34 SuzuKi, K., PODUSLO,S. E., AND NORTON,W. T., Gangliosides in the myelin fraction of developing rats, Biochim. biophys. Acta (Amst.), 144 (1967) 376-381. 35 VANCE,n . E., AND SWEELEY,C. C., Quantitative determination of the neutral glycosyl ceramides in human blood, d. Lipid Res., 8 (1967) 621-630. 36 VIRCHOW,R., Ueber das ausgebreitete Vorkommen einer dem Nervenmark analogen Substanz in den Thierische Gewebe, Virchows Arch. path. Anat., 6 (1854) 562-572. 37 WALLACH,D. F. H., Isolation of plasma membranes of animal cells. In B. D. DAvis AND L. WARREN (Eds.), The Specificity of Cell Surfaces, Prentice-Hall, Englewood Cliffs, N.J., 1967, pp. 129-141. 38 WOLMAN,M., Myelin breakdown in vitro, Biochim. biophys. Acta (Amst.), 102 (1965) 261-268. 39 WORTHINGTON,C. R., ANDBLAUROCK,A. E., Low-angle X-ray diffraction patterns from a variety of myelinated nerves, Biochim. biophys. Acta (Amst.), 173 (1969) 427--435. 40 WRIGGLESWORTH,J. M., AND PACKER, L., pH-Dependent conformational changes in submitochondrial particles, Arch. Biochem., 133 (1969) 194-197.
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
NON-OSMOTIC S W E L L I N G IN P U R I F I E D BOVINE M Y E L I N
D. L. MclLWAIN* Division of Neuroscience, New York State Psychiatric Institute, New York, N.Y. 10032 (U.S.A.)
(Accepted August 31st, 1972)
SUMMARY Purified, lyophilized bovine myelin in aqueous susFension exhibits non-osmotic swelling which is influenced by the pH, ionic strength and temFerature of the bulk medium. Trypsin, chymotrypsin, papain or pronase can initiate swelling, as observed by phase contrast microscopy and turbidimetry. Swollen myelin is mechanically fragile and forms protein-deficient vesicles, some of which are not sedimented in water at 100,000 × g for 1 h. The swelling process is similar to changes reported for myelinated nerves in several experimental and pathological states and resembles two types of swelling known to occur in unilamellar membranous structures.
INTRODUCTION Swelling of the myelin sheath, accompanied by lamellar splitting and fragmentation, has been observed in demyelinating diseases 19,3a and their experimental analogues 3,27. It is not clear, however, whether these effects are directly or indirectly produced by the toxic agents used experimentally, nor are the underlying molecular changes well understood. X-ray diffraction11, a9 and electron microscopic 28 studies have demonstrated that normal myelinated nerve fibers swell in media of low ionic strength. Both techniques indicate an accumulation of water at the intraFeriod line of myelin, with splitting of the concentrically-arranged membrane pairs. Worthington and Blaurock 39 believe this type of swelling to be non-osmotic, since it can occur in hypertonic sucrose solutions and is inhibited by low concentrations of salts of divalent cations, and these authors raise the possibility that a structural change occurs within the membrane pairs during swelling. * Present address: Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N.C. 27514, U.S.A.
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Studies on purified membrane should permit one to investigate the direct efl'ects of swelling agents on myelin. However, a difficulty arises in the use of fresh myelin preparations for studying the swelling phenomenon. As one might expect from the electron microscopic and X-ray diffraction data cited above 11,2s,ag, myelin swells during isolation by the usual techniques in low ionic strength media. When examined by phase microscopy, myelin freshly prepared by the procedure used here shows very low refractivity, unlike the highly refractile membrane in vivo or in salt solutions 7. This change can be correlated with the swelling of myelin (see below). A recent report by De Vries et al. 7 indicates that swelling of myelin occurs within 1-2 h in ice-cold media of low ionic strength and is relatively unaffected by the osmolarity of sucrose in the medium. On the other hand, myelin which has been lyophilized and then rehydrated regains its highly refractile appearance and, more importantly, mimics a number of the features of myelin undergoing swelling and fragmentation in vivo or in isolated or cultured myelinated nerve fibers. The present study describes the swelling effects of several agents on rehydrated myelin preparations. A clearer understanding of the membrane changes described here may provide useful information not only about the nature of myelin swelling and degradation in the demyelinating diseases, but also about the molecular organization of normal myelin. MATERIALS AND METHODS
Lyophilized myelin was prepared from bovine pons and medulla by the method of Suzuki et a l ) 4 and stored at --20 °C. Lipids were extracted from myelin (12.4 mg dry weight) after first dissolving it in 1 ml chloroform-methanol (2:1) and evaporating to dryness 3 times, causing approximately 75 % of the protein to adhere to the test tube. The chloroform-methanol (2:1) soluble material was then placed on a silicic acid column (1 g) and eluted with 12 ml methanol. Quantitative yields of myelin lipid were obtained, while 92 % of the protein, as measured by the method of Lowry et al. z° was removed by this technique. Trypsin (crystallized × 2), chymotrypsin (crystallized × 3), phospholipase C (C1. perfringens) and galactose oxidase were obtained from Worthington Biochemical Corp., Freehold, N. J. ; pronase (B grade) and papain (african papaya), from Calbiochem, Los Angeles, Calif.; soybean trypsin inhibitor from Armour Laboratories, Chicago, 111.; ovomucoid (chicken) trypsin inhibitor, a-N-benzoyl-D,L-arginine-pnitroanilide HC1 and Dextran 15 (average mol. wt., 20,000), from Sigma Chemical Co., St. Louis, Mo. Diphenylcarbamylfluoride was a gift from Dr. Norbert Wassermann. Dextran 10 (number average mol. wt., 5,700) was purchased from Pharmacia Fine Chemicals, Inc., Piscataway, N.J. Phase contrast microscopy
Freeze-dried myelin suspended by gentle (bath type) sonication in water (2 mg/ml) and combined with an equal volume of the appropriate test solution was placed between two plastic coverslips, resting on a glass slide, and viewed with a Leitz Ortholux phase contrast microscope. Plastic coverslips were used to avoid pH
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increases reported in aqueous films between glass surfaces 26. Coverslip effects, including increased myelin swelling, were nonetheless noted in samples which were allowed to stand for over 15 min. Thus, specimens were viewed and photographed no later than 5 min after mounting. Myelin lipids (10/~g) were dried on a glass slide and viewed under low power after introducing a drop of the appropriate buffer onto the sample and covering it with a glass coverslip. By using buffered systems and comparing the results obtained with and without a coverslip, artifacts arising from pH alterations were avoided.
Turbidimetry Optical density measurements of myelin suspensions were carried out at 520 nm, using a Beckman Model B spectrophotometer. Myelin (2 mg/ml) was suspended in deionized water by gentle sonication, and the O.D. recorded 15 min after adding an equal volume of a solution of appropriate pH and ionic strength. The pH of the sample was measured immediately after determining the O.D.
Gas-liquid chromatography ( GL C) Methyl esters of free fatty acids were prepared by treating myelin lipids (750 #g) with diazomethane in 0.5 ml cold methanol--diethylether (1:9). Palmitic acid was included as an internal standard. Methyl esters were injected onto a 15 ~ EGSS-X on 100/120 GasChrom P column (Applied Science Labs., State College, Pa.), using an Aerograph Hi-Fy apparatus (hydrogen flame ionization detector) operated isothermally at 180 °C. Peak areas were measured by multiplying the height times the width at one-half peak height. Galactose was measured quantitatively by GLC, using the procedure of Vance and Sweeley35. A 3 ~ OV-1 100/200 GasChrom Q glass column at 160 °C was employed, with mannitol as an internal standard. Quantitative thin-layer chromatography was used to separate myelin phospholipids, which were assayed for P as described by Mcllwain and Rapport 22. Cholesterol was estimated by the method of Schoenheimer and Sperry 30 and total reducing sugar by the phenol-sulfuric acid assay 17. RESULTS
Types of swelling Purified myelin, suspended by sonication in aqueous media, formed multilamellar particles 1-10 #m in diameter 22 which then aggregated to produce clumps visible by phase contrast microscopy (Fig. 1). Changing the pH of the suspension not only altered the degree of clumping, but also caused the particles to swell in many instances. Two morphologically different types of swelling were observed: (1) a roughly symmetrical form at pH < 3 or > 8 (Fig. 2) and (2) evaginative forms at pH 6-8, appearing as bulbous or elongated outgrowths from the myelin (Fig. 3). The former involved all myelin particles, while the latter did not always do so. Symmetrical swelling was accompanied by an apparent decrease in the refractivity of the fragments, giving them a dark grey appearance by phase microscopy, while the evaginations
Fig. 1. Phase contrast appearance of myelin suspended in water. Freeze-dried myelin was sonicated in deionized water (I mg/ml) and viewed between plastic coverslips. 500.
Fig. 2. Symmetrically swollen myelin. Same sample as in Fig. I, immediately after addition of 10/d of 0.1 N HC1 between coverslips. , 500.
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Fig. 3. Evaginative swelling in myelin. Freeze-dried membrane was sonicated in water (1 mg/ml) and diluted with an equal volume of 20 m M Tris-HC1 buffer (pH 8.1). Phase contrast micrographs were made of samples 30 min after preparation (25 °C). x 500.
often arose from highly refractile clusters of myelin and in the presence of salts (see below) sometimes appeared thick-walled and refractile themselves. The elongated, grey evaginations were mobile and fragile, often pinching off spontaneously to form free-floating vesicles. The long evaginations were never observed arising from symmetrically swollen particles.
Effects of pH The relationship between the pH of the medium and the phase contrast appearance of dispersed myelin in the absence of added salts can be summarized as follows: at pH < 3 the myelin particles were well dispersed and exhibited symmetrical, but not evaginative swelling. Between pH 3 and 4.5 clumping increased markedly, with no sign of swelling of the particles. Clumping decreased from pH 4.5 to 7.0 and elongated forms were noted between pH 6.0 and 8.0. The evaginations appeared within 5 rain at pH 7.4 and increased in size and number over a period of several hours. At pH 8-11, symmetrical swelling was rapid and obvious, although in the presence of salts, evaginations also occcurred above pH 8. The refractivity of the myelin particles decreased above pH 8.0, and above pH 10 many myelin particles disintegrated into very small, grey vesicles, which floated in the medium.
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pH Fig. 4. Relationship between the pH and optical density (520 rim) of aqueous myelin suspensions. Myelin was suspended in water and adjusted to the desired pH with dilute HC1 or NaOH to give membrane concentrations of 0.125 mg/ml (r-7 17); 0.25 mg/ml (O O); and 0.50 mg/ml (~ A). The O.D. is expressed relative to that for a like concentration of myelin in 8 mM imidazole-HC1 buffer at pH 7.0. Each point represents one sample. Closed circles ( 0 - - - - 0 ) denote samples of myelin (0.25 mg/ml) suspended in 2 mM CaCI2, final concentration, and adjusted to the indicated pH.
These changes were correlated with alterations in the O.D. of the suspension at 520 nm (Fig. 4). Samples containing different concentrations of myelin were adjusted to p H values from 2 to 11. A decrease in O.D. would be expected either with an increase in clumping of the particles or with swelling of the myelin. Decreases in O.D. based on particle collisions (clumping) should vary as a function of the myelin concentration, unlike swelling 4°. As can be seen (Fig. 4), differences in the percent decreases in O.D. of the 3 suspensions occurred only between p H 3 and 7, the range in which clumping was observed microscopically. Below p H 3 and above p H 7, however, these differences disappeared, indicating that decreases in O.D. in these ranges reflected swelling of the myelin particles. The two types of swelling could not be distinguished by the O.D. changes shown in Fig. 4. However, the rapid decrease in O.D. above p H 9.5 appeared to correlate with the onset of disintegration of the symmetrically swollen myelin. The decrease in turbidity of myelin suspension at pH 9 was partially reversed by back titration with dilute HC1, while at p H 2-3 the O.D. did not increase with N a O H addition. Myelin preparations which did not disperse well in water gave similar, but somewhat flatter curves than shown in Fig. 4. Less than 1.6/zg of free fatty acids and aldehydes per mg dry myelin were detected by G L C of lipid extracts from suspensions previously adjusted to p H 1.9 with HCI
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or to pH 10.5 with NaOH (and in control samples at pH 7.1) and then extracted with 4 vol. of chloroform-methanol (2 :I) at pH 4.0. If liberated during this procedure, these long-chain products would account for the cleavage of less than 2 % of acid and alkali labile phosphatides in myelin. Moreover, no changes in phosphatide composition were found by quantitative TLC of myelin exposed to the same extremes of pH and compared to control samples at pH 7.1.
Salt effects The presence of salts affected aqueous suspensions of myelin in several ways. First, the salts of monovalent and divalent cations promoted clumping of the particles. This effect was clearly seen with 8 mM imidazole-HC1 buffers (pH 7.0) containing 100 mM NaC1, 100 mM KCI, 5 mM CaCI2 or 5 mM MgCI2. Secondly, these salts retarded swelling at alkaline pH. For example, inhibition of swelling was observed with 5 mM CaC12 or MgC12 and with 1 M NaCI or KC1 between pH 7.0 and pH 9.0. NaC1 and KCI (100 mM) were much less effective in this regard than 5 mM CaC12 or MgC12 at pH 9.0. Thirdly, in certain instances salts influenced the morphology of swollen myelin. In 0.5 mM CaC12 at pH 8.0 highly refractile evaginations were often produced, unlike the grey ones seen in the absence of CaC12. Moreover, evaginative, rather than symmetrical swelling was observed between pH 8 and 11 when 5 mM CaCI2 was present. The O.D. changes in myelin suspensions containing 2 mM CaC12 and adjusted to various pH values are shown in Fig. 4. It can be seen that 2 mM CaCI~ retarded swelling between pH 7 and 11 much more than at low pH and promoted clumping between pH 3 and 7.
Time and temperature effects The temperature of the myelin suspension during incubation also affected the degree of swelling visible by phase microscopy. When myelin (1 mg/ml) was suspended in 8 mM imidazole-HC1 (pH 7.4) at 5 °C and aliquots were taken for subsequent incubation at 5 °C, 37 °C and 90 °C for 10 rain, increases in evaginative swelling varied directly with the temperature: while little apparent swelling took place at 5 °C, many long, thin evaginations were observed at 90 °C, with a moderate number appearing at 37 °C. In addition, swelling slowly increased with time in these samples. As expected, the O.D. of such suspensions decreased with increasing time and temperature, but this was found (by varying the myelin concentration) to be caused, in part, by increased clumping of myelin. Aqueous suspensions of myelin at 5 °C showed decreases in O.D. equal to those seen at 25 °C (Fig. 4) as their pH was increased from 7 to 9. That swelling occurred at such extremes of temperature and pH militated against an enzymatic basis for the process.
Swelling of myelin lipids The morphology of evaginative forms of swelling already described was strikingly similar to 'myelin forms' known to arise from a variety of lipid emulsions. In order to compare the swelling of myelin lipids with that of the parent membrane, myelin
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lipids were exposed to media of varying pH and ionic strength. In deionized water, tubular formations appeared almost instantly and grew exuberantly under the coverslip for several minutes. The forms resembled those of myelin membrane suspensions in several ways: they presented a large variety of elongated and bulbous shapes, had the same grey hue of the membrane evaginations, were highly mobile and capable of spontaneously vesiculating. Furthermore, their appearance was retarded by the presence of salts in the medium. Solutions containing l0 mM CaCI2 or MgCI2 markedly inhibited the development of such forms, while as little as 1 mM CaCl2 substantially reduced the swelling. NaCI and KC1 (0.5-1 M) showed inhibitory effects comparable to those to 5 mM CaCI2. Moreover, these salts altered the morphology of the orms, making them shorter, thinner and more refractile, as they did evaginations from the membranes. Besides their more rapid swelling, myelin li~'ids difJered from the parent membranes in their responses of pH changes. In media of low ionic strength myelin lipids exhibited swelling which did not vary noticeably between pH 3 and 11. Two different aqueous s y s t e m s - citric acid-Na2HPO4 buffers (10-15 mM); and glycine-HCl, NaOAc, and imidazole-HC1 buffers (10 raM) --were employed in these experiments. In contrast, 20 mM HC1 (pH 1.8) retarded swelling of the liFids, lzroducing highly refractile, slowly develoring forms.
F~{/bct of proteases The differences between myelin lipids and membranes with respect to pH effects on swelling suggested that myelin proteins played a role in the phenomenon. Much stronger evidence for this p~ssibility was obtained in experiments with several proteolytic enzymes. Myelin (1 rag) suspended in I ml of 8 mM imidazole-HCl buffer at pH 7.0, containing 20 #g of trypsin/ml, showed marked evaginative swelling within 10 rain at 25 °C (Fig. 5), whereas samples containing no enzyme or enzyme preincubated with one molar equivalent of soybean trypsin inhibitor were only slightly swollen. Trypsin-induced swelling was inhibited by 5 mM CaCI2, but not by 2 mM Dextran 10. As little as 1 #g of trypsin/ml produced evaginations under these conditions. A much greater decrease in O.D. occurred in myelin suspensions exposed to trypsin than in samples with soybean trypsin inhibitor (STI) or no enzyme (Fig. 6). One to 11 molar equivalents of diphenylcarbamylfluoride(DPCF) 9, preincubated with trypsin for 30 min at 25 °C, caused 80-90% inhibition of the initial rate of myelin swelling and inhibited the hydrolysis by trypsin of fl-N-benzoyl-D,L-arginine-pnitroanilide-HCl1° to a comparable extent. On the other hand, 1-10 molar equivalents of ovomucoid trypsin inhibitor (OTI) reduced the initial rate of myelin swelling 70-80 %, while equimolar quantities of the inhibitor caused 98 % inhibition of trypsin hydrolysis of the aforementioned synthetic substrate. No swelling or clumping resulted from exposure of myelin to OTI alone. Turbidimetric and microscopic effects very similar to those above were obtained with identical concentrations of chymotrypsin (pH 7.0), pronase (pH 7.0) and papain (pH 6.5). Heating chymotrypsin and pronase at 100 °C for 10 rain reduced the initial rate of swelling to 20 ~o or less of that seen with the untreated enzymes, as did exposure
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NON-OSMOTIC SWELLING IN MYELIN
Fig. 5. Phase contrast micrograph of trypsin-treated myelin. Membrane was suspended i n water and diluted with imidazole-HCl buffer (pH 7.0) to give 1 mg myelin per ml 8 m M buffer. The sample was then exposed to trypsin (20 #g/ml) for 10 min at 25 °C. × 500.
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Fig. 6. Trypsin-induced decrease in turbidity of myelin suspensions. Membrane was suspended in 8 m M imidazole-HC1 buffer (pH 7.0) as in previous figure. Trypsin (20/~g/ml, final concentration) was added and the O.D. changes were recorded at 1 min intervals. Trypsin plus myelin ((3 ©); trypsin preincubated with equimolar STI for 15 min at 25 °C and added to myelin suspensions (20 Fg enzyme/ml) (O Q); myelin without enzyme ([] []).
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of chymotrypsin to one molar equivalent of DPCF. The papain effect (20 big enzyme/ mg myelin/ml) was completely eliminated by 100/zM p-chloromercuribenzoic acid (PCMB). When used at the above concentrations, D P C F and PCMB did not inhibit the swelling process directly, since they showed no inhibition ofpronase-induced swelling. Phospholipase C (CI. perfringens), which can cleave 64~o of the myelin phospholipids 22, did not cause swelling under similar conditions. The question arises as to whether an osmotic mechanism could account for the swelling produced by the above agents. Osmotically active molecules previously trapped within the myelin particles could not be responsible, since their effects would have been exerted immediately upon suspension of myelin in water, before addition of the swelling agent. Donnan effects are also not likely, because swelling was induced by raising the temperature of myelin suspended in distilled water only. On the other hand, it is possible that the agents release membrane protein into the intralamellar spaces, causing water to enter, separate the membranes and increase the volume of the particles. The data on fragmentation of swollen myelin (see below) make this a plausible explanation. In order to test this, the maximum concentration of protein available for release (250 #g/mg dry myelin; predominantly 18,000-34,000 tool. wt.) was estimated by assuming that all the protein is released into the aqueous volume represented by the difference between the water space before and alter swelling. This volume difference was determined gravimetrically on pellets from suspensions of myelin at p H 6.8 and 10.8 and found to be 6.5 ~ 0.4/zl/mg dry myelin, representing a 250~,, increase in volume during swelling. A maximum concentration of approximately 1 m M protein was then calculated. Using this figure, an attempt was made to reverse swelling osmotically with a non-penetrating molecule added to the swollen myelin at comparable concentrations. Table I shows the results of such experiments. When 2 m M (final concentration) Dextran 10 (number average tool. wt. 5,700) was added to myelin swollen at pH 10.1, no reversal of swelling was noted. Similar negative results were obtained with the larger Dextran 15 (average mol. wt. 20,000). The situation was different at pH 2, however, where both dextran solutions, particularly Dextran 15,
TABLE I EFFECT OF DEXTRANS ON MYELIN SWELLING
Myelin (1 rag) was suspended in 1 ml water and diluted with an equal volume of 17 mM imidazoleHCI buffer (pH 7.0), 17 mM Na~B407 buffer (pH 10.3-10.5) or 0.02 N HCI. After mixing, 1 ml of the appropriate buffer, with or without 4 mM Dextran 10 or 15, was added. The final pH was then measured and the O.D. at 520 nm recorded. Percent decrease in O.D. is given relative to the O.D. of the suspensions at pH 7.0. After swelling, added
Buffer only Buffer + Dextran 10 Buffer ÷ Dextran 15
% Decrease in O.D. at: pH 2.0
pH 10.1
40.4 ± 5.8 (S.D.) 33.4 ± 4.0 14.6 ± 4,7
29.6 4- 2.3 35.9 ± 2.2 33.5 ± 5.2
NON-OSMOTIC SWELLING IN MYELIN
107
Fig. 7. Electron microscopic appearance of membranous fragments which did not sediment in water at 56,500 x g for 15 min. The sample was prepared by a two-step centrifugation technique from myelin swollen at 38 °C for 1 h in 6 mM imidazole-HCl buffer (pH 7.4) containing 2.2 mM CaCI2. After sedimentation at 56,500 x g for 15 min, the pellet was suspended in water by sonication and recentrifuged at 56,500 x g for 15 rain. The vesicles, found in the second supernatant fraction, were sedimented in 20 mM CaCI2 and the pellet was then fixed in 3 ~ glutaraldehyde (pH 7.4) and stained with uranyl acetate-lead citrate, x 26,300. caused a reversal o f the relative O.D. Decreasing the p H o f the d e x t r a n - c o n t a i n i n g solutions to p H 1.7 d i m i n i s h e d the O . D . and p r o d u c e d symmetrically swollen particles similar to those p r e s e n t before a d d i t i o n o f the polysaccharide. Thus, swelling at acid p H c o u l d p r o c e e d in the presence o f the Dextran, b u t r e q u i r e d a lower p H t h a n in the absence o f the D e x t r a n . The reason for this change is n o t clear. In a n y case, the results indicate t h a t swelling o f myelin at b o t h p H extremes is largely a n o n - o s m o t i c
108
I). k . M C I L W A I N
process. The lack of effect of 2 mM Dextran 10 on trypsin-induced myelin swelling (see above) further supports such an interpretation.
Fragmentation of swollen myelin Myelin exhibiting evaginative or symmetrical swelling could vesiculate spontaneously. Phase contrast microscopy showed that sonication of these swollen preparations markedly increased vesiculation and reduced the size of the fragments formed. The vesicles, which could be produced from myelin swollen under quite mild conditions, were separated from the remaining myelin particles by centrifuging the sonicated preparation in water. For example, myelin showed moderate swelling when incubated at 38 °C for 1 h in 6 mM imidazole-HCl buffer (pH 7.4) containing 2.2 m M CaCl2 (ef. ref. 22, Fig. 2). The sample was then sedimented at 56,500 × g for 15 rain and the supernatant fraction removed. Upon sonication of the pellet in water and recentrifugation at 56,500 × g for 15 rain, vesicles approximately 0. l-0.5/zm in diameter (Fig. 7) were separated from the large particles. The vesicles, which were precipitated with 10 m M CaC12 before electron microscopy, appeared to exist, as such, before addition of the salt: 70 ~o were removed from water by passing the suspension through a cellulose acetate (Millipore) filter with an average pore diameter of 0.22/~m, while 39 ~o would not pass a filter with an average pore diameter of 0.45 /~m. When the filters were soaked in such suspensions for 1 h, 11 ~o of the fragments adhered to them, indicating some non-specific binding. The relationship between swelling and vesiculation was studied by the two-step centrifugation technique employed above, which allowed all samples of swollen myelin to be sonicated and centrifuged in the same medium (water). The quantity of non-sedimented phospholipid in supernatant 2 (Table II) was directly related to the p H at the time of swelling, when studied between pH 6.8 and 11.2. The percent total
TABLE II ANALYSIS OF NON-SEDIMENTABLE MYELIN PHOSPHOLIPID AND PROTEIN
Myelin suspensions at designated pH were centrifuged at 56,500 × g for 15 min (5 °C). Pellets were resuspended by sonication in water and centrifuged a second time at 56,500 x g for 15 min. In sample 7, myelin suspensions in 8 mM imidazole-HC1 (1 mg/ml) were first treated with trypsin (20 ttg/ml) at 25 °C for 30 rain.
Sample No.
1 2 3 4 5 6 7
deionized water, aqueous NaOH, aqueous NaOH, aqueous NaOH, aqueous NaOH, aqueous NaOH, trypsin (20 #g/ml),
Supernatant 1
Supernatant 2
°/o total phospholipid
Protein/P % total Protein/P (w/w) phospholipid (w/w)
pH 6.8 12.4 ± 0 pH 7.1 15.0 ~ 0.7 pH 7.9 14.8 ± 0.6 pH 9.6 13.2 ~- 1.0 pH 10.5 18.8 ± 4.5 pH 11.2 14.9 ~ 0.3 pH 7.0 13.0 ± 1.2
3.5 5.0 8.5 10.6 14.4 25.0 --
6.2 ± 8.7 ± 15.0 i 25.2 ~ 28.2 ± 33.6 ± 31.0 ±
1.3 0.6 0.01 1.1 3.0 3.9 2.9
11.2 3.8 8.1 7.5 10.7 13.3 6.7
NON-OSMOTIC SWELLING IN MYELIN
109
phospholipid in the first supernatant was approximately the same for each sample and probably represented fragments originally present in the myelin preparations. Even when centrifugation was performed at I00,000 × g for 1 h, 10-15~ of the total phospholipid still remained in supernatant 2, after swelling at pH 10.7 or under conditions used to produce the fragments shown in Fig. 7. The protein :phosphorus ratio in freeze-dried myelin was 16:1. It can be seen (Table II) that all the supernatant fractions, particularly those samples between pH 7 and 10, were deficient in protein, except sample 6, supernatant 1. Vesicles of similar composition were isolated from myelin swollen by treatment with trypsin (sample 7, Table II). The protein:P ratio in supernatant 1 increased as the pH was raised, possibly indicating solubilization of protein at higher pH values. A similar pattern was observed in supernatant 2, except between pH 6.8 and 7.1. The initial protein content was completely accounted for in the supernatant and pellet fractions. Phospholipid: galactolipid : cholesterol ratios in the vesicles and in pellet fractions from samples treated as that in Fig. 7 or swollen at pH 10.7 were similar to the ratios found in lyophilized myelin (1.9:1.1:1.0). DISCUSSION
Evidence supporting these major points has been presented: (1) isolated myelin can swell by other than an osmotic mechanism; (2) two forms of swelling were noted, and (3) both were pH-dependent; (4) proteases and elevated temperature enhanced swelling, while salts were inhibitory at the concentrations studied; (5) the process could be monitored quantitatively by turbidimetry; (6) swollen myelin was mechanically fragile, forming protein-deficient vesicles which were not easily sedimented by high-speed centrifugation. The similarity in the effects of swelling agents on rehydrated myelin to those reported for intact myelin and a number of other membranes, discussed below, strongly suggests a common mechanism of swelling. Swelling in purified myelin resembled that of intact medullated fibers in being non-osmotic 89, sensitive to salts, especially those of divalent cations 3s, and to trypsin ~. The decrease in refractivity of myelin observed by Masurovsky and Bunge 21 during swelling of cyanide-poisoned, cultured peripheral nerve fibers is strikingly similar to that described here. In view of the marked alkalinizing effect of NaCN, it is possible that at the concentration of NaCN (10 mM) used in that study a pH effect was superimposed on the expected metabolic poisoning. Whether the two processes are identical must await a complete ultrastructural analysis of the changes in purified myelin and a detailed comparison of the effects on each by the swelling agents described here. Swelling and fragmentation of myelin have been reported in multiple sclerosis lesions 33. In a series of histologic investigations Hallpike, Adams and Bayliss have demonstrated an increase in proteolytic activity in demyelinating tissue in multiple sclerosis 14 and Wallerian degeneration15. They have also described the early appearance in degenerating nerve oI 'myelin buds', presumably similar to the evaginations shown here, which they attributed to increased proteolytic activity in the tissue 16.
1 IO
D.L. MCILWAIN
Moreover, fusiform swelling of myelin and subsequent fragmentation were prominent features in cultured myelinated nerve fibers exposed to antisera from rabbits with experimental allergic encephalitis a, sera from patients with multiple sclerosis is, sera containing antigalactocerebroside antibodies 8, and to several other demyelinating agents 5,~12-5. It is reasonable that the swelling process described here could be related to these pathological situations and may provide a useful way to discriminate between direct and indirect effects of these toxic substances on myelin. Similar forms of swelling are known to occur in structures bounded by a single membrane, and these are apparently initiated by changes within the membrane itself. One general class may involve a change in the conformation of membrane protein leading to alteration of membrane size and shape. Examples of this type include the Ca2+-activated contraction of erythrocyte ghosts 24 and the pH-dependent swelling of submitochondrial particles 40. Using optical rotatory dispersion and light-scattering techniques in the latter study, Wrigglesworth and Packer attributed increases in size of the unilamellar particles at high pH to disaggregation of membrane protein. A relationship between the mechanism of swelling in that system and in myelin is suggested by the similarity of the light-scattering-pH profile of submitochondrial particles to the O.D.-pH profile of myelin (Fig. 4). That changes in myelin protein were implicated in the swelling process may also be inferred from the differences in swelling of myelin lipids and myelin membranes - - the former occurring more rapidly and largely independent of pH - - and, more directly, from the fact that proteolytic enzymes also produced evaginations. Incomplete inhibition of trypsin-induced swelling by OT1 and DPCF left open the possibility that hydrolysis of protein was not required for the effect. However, it is noteworthy that myelin basic and acidic proteins, but not proteolipid protein, are susceptible to cleavage by trypsin la. Other factors (temperature, salts) shown here to influence swelling also may have exerted their effects, at least in part, on myelin protein. The protein possibly inhibited the swelling process in some way, and an alteration in myelin protein may have been an early event in the changes noted. Another similar type of swelling in biological membranes resembles the wellknown 'myelin forms' arising from lipid emulsions. Evaginations, termed 'stromatolytic forms', have been produced from erythrocyte ghosts under conditions where swelling occurred against or without an apparent osmotic gradient 12. Furchgott suggested that the molecular changes occurring within the ghost membrane involved a reorganization of lipid and possibly protein lz. Similar forms have been observed arising from a number of tissues and cells, including myelinated nerve fibers 12,a6, grey and white matter 6,al, liver slices 2a, erythrocytes ~,2, and leukocyteszg. In the present study the evaginations were like 'myelin forms' produced from suspensions of myelin lipids both morphologically and in their response to salts. The relationship of evaginative to symmetrical swelling and their comparative effects on the turbidity of the suspensions remain undefined, although the two types of swelling may represent different phases of the same process, with evaginations resulting earlier from focal changes in the membrane. What then can be said with regard to the mechanism of swelling in myelin? It
NON-OSMOTIC SWELLINGIN MYELIN
111
seems probable that changes in the organization of both myelin protein and lipid accompany swelling, with alterations in protein possibly initiating the process. Moreover, the marked decrease in protein: P ratios in membrane fragments which were swollen at high pH or by exposure to trypsin indicates that the molecular disruption was extensive. However, in only one instance (protease treatment) was there reason to suspect cleavage of covalent bonds during swelling. The question then arises as to whether these changes represent a rearrangement of the myelin layers (e.g. an unrolling or fusion of the lamellae) or an actual increase in the surface area of the membrane. In the case of the unilamellar submitochondrial particles 4° surface area expansion seemed to occur at pH 9. This suggests a like mechanism for myelin. However, the answer must await more direct experimental support. Since the O.D. of myelin suspensions varied as a result of effects of pH and ionic strength on components of the membrane, there is reason for caution in the use of tubidimetry (or other light-scattering methods) for measuring osmotically-induced volume changes in membranous structures. A purely passive role for the membranes of myelin, submitochondrial particles and possibly other structures in osmotic gradients and at different pH values cannot be safely assumed. That membranes fragment in media of low ionic strength or high pH is well known 37, although the details of the process are not. In the present study fragmentation of myelin was correlated with pH- and protease-induced swelling of the membrane. It would be of interest to know if fragmentation of other membranes (e.g. red cell ghosts 4,a2) under similar conditions is also preceded by swelling. The decrease in both size and protein content of the vesicles produced from swollen myelin probably accounted for their not being sedimented under the conditions described here. Such a process could easily lead to contamination of supernatant fractions in brain homogenates, since many of the myelin fragments were not sedimented in water at 100,000 × g for 1 h. Moreover, the fact that membranous structures appeared in these high-speed supernatant fractions underlines the ambiguity of the term 'soluble' frequently applied to such fractions. It will, therefore, be of considerable importance to determine whether similar processes actually occur during preparation of myelin or other biological membranes in media of low ionic strength, such as sucrose. ACKNOWLEDGEMENTS The author thanks Dr. M. M. Rapport for his support of this study and Dr. Leon Roizin and Mr. Jevons Liu for performing the electron microscopy.
REFERENCES 1 BAKER,R. F., The fine structure of stromatolytic forms produced by osmotic hemolysis of red blood cells, J. Ultrastruct. Res., 11 (1964)494-507. 2 BESSIS,M., Phase contrast microscopy and electron microscopy applied to blood cells, Blood, 10 (1955) 272-286. 3 BORNSTEIN,M. B., AND APPEL, S. H., Tissue culture studies of demyelination, Ann. N.Y. Acad. Sci., 122 (1965) 280--286.
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4 BRAMLEY,Y. A., COLEMAN,R., AND FINEAN,J. B., Properties oferythrocyte membranes, Biochim. biophys. Acta (Amst.), 241 (1971) 752--769, 5 BUNGE, R. P., Myelin degeneration in tissue culture, Neurosei. Res. Progr. Bull., 9 (1971) 496~498. 6 CASPER,J., AND WOLMAN, M., Demonstration of myelin figures by fluorescence in some pathological tissues, Lab. Invest., 13 (1964) 27-31. 7 DE VINES, G. H., NORTON, W. T., AND RAINE, C. S., Axons: Isolation from mammalian central nervous system, Science, 175 (1972) 1370-1372. 8 DUBOIS-DALCQ,M., NIEDIECK, B., AND BUVSE, M,, Action of anti-cerebroside sera on myelinated nervous tissue cultures, Path. Europ., 5 (1970) 331-347. 9 ERLANGER, B. F., AND COHEN, W., Specific inactivation of chymotrypsin by diphenylcarbamyl chloride, J. Amer. chem. Soc., 85 (1963) 348-349. 10 ERLANGER, B. F., KOKOWSKY, N., AND COHEN, W., The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem., 95 (1961) 271-278. 11 FINEAN, J. B., AND MILLINGTON, P. F,, Effects of ionic strength of immersion medium on the structure of peripheral nerve myelin, J. biophys, biochem. Cytol., 3 (1957) 89-94. 12 FURCHGOTT,R. F., Observations on the structure of red cell ghosts, Cold Spring Harbor Syrup. Quant. Biol., 8 (1940) 224-232. 13 GONZALES-SATRE, F., The protein composition of isolated myelin, J. Neurochem., 17 (1970) 1049-1056. 14 HALLPIKE, J. F., ADAMS, C. M. W., AND BAYLISS, O. B., Proteolytic activity around multiple sclerosis plaques, Histochem. J., 2 (1970) 199-208. 15 HALLPIKE, J. F., ADAMS, C. M. W,, AND BAYLISS,O. B., Neutral and acid proteinases in early Wallerian degeneration, Histochem. J., 2 (1970) 209-218. 16 HALLPIKE,J. E., ADAMS,C. M. W., AND BAYLISS,O. B., Proteolysis of normal myelin and release of lipids by extracts of degenerating nerve, Histochem. J., 2 (1970) 315-321. 17 HAMILTON,J. K., REBERS,P. A., DUBOIS, M., GILLES, K. A., AND SMITH, F., Colorimetric method for determination of sugars and related substances, Analyt. Chem., 28 (1956) 350-356. 18 KIM, S. U., MURRAY, M. R., TOURTELLOTTE,W. W., AND PARKER,J. A., Demonstration in tissue culture of myelinotoxicity in cerebrospinal fluid and brain extracts from multiple sclerosis patients, J. Neuropath. exp. Neurol., 29 (1970) 420-431. 19 LAMPERT, P. W., Fine structural changes of myelin sheaths in the central nervous system. In G. H. BOURNE(Ed.), The Structure and Function of Nervous Tissue, Vol. I, Academic Press, New York, 1968, pp. 187-204. 20 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL,R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 21 MASUROVSKY,E. B., AND BUNGE, R. P., Patterns of myelin degeneration following the rapid death of cells in cultures of peripheral nervous tissue, J. Neuropath. exp. Neurol., 30 (1971) 311-324. 22 MCILWAIN, D. L., AND RAPPORT, M. M., The effects of phospholipase C (Cl. perfringens) on purified myelin, Biochim. biophys. Acta (Amst.), 239 (1971) 71-80. 23 PALADE,G. E.. AND CLAUDE,A., The nature of the Golgi apparatus, J. Morphol., 85 (1949) 35-69. 24 PALEK,J.,CURIIY,W.A.,AND LtONETTI,F.J.,Calcium ATPaseandcalcium-linkedghost shrinkage, Amer. J. Physiol., 220 (1971) 1028-1032. 25 PETERSON,E. R., AND MURRAY, M. R., Patterns of peripheral demyelination in vitro, Ann. N. Y. Acad. Sei., 122 (1965) 39-50. 26 PONDER, E., Hemolysis and Related Phenomena, Grune and Stratton, New York, 1948, p. 41. 27 RAINE, C. S., AND BORNSTEIN,M. B., Experimental allergic encephalomyelitis: an ultrastructural study of experimental demyelination in vitro, J. Neuropath. exp. Neurol., 29 (1970) 177-191. 28 ROBERTSON,J. D., Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions, J. biophys, biochem. Cytol., 4 (1958) 349-364. 29 ROZENSZAJN,L. A., EPSTEIN, Y., AND FISCHER, D., Myelin figures in blood cells, lysosomes and lipidoses, Amer. J. clot. Path., 52 (1969) 473-476. 30 SCHOENHEIMER,R., AND SPERRY, W. M., A micromethod for the determination of free and combined cholesterol, J. biol. Chem., 106 (1934) 745-760. 31 SHANKLIN,W. M,, Myelin figures demonstrated with the phase microscope, Stain Teehnol., 36 (1961) 377-378. 32 STECK,T. L., WEINSTEIN,R. S., STRAUSS,J. H., AND WALLACH,D. F. H., Inside-out redcell membrane vesicles: preparation and purification, Science, 168 (1971) 255-257. 33 SUZUKI,K., ANDREWS,J. M., WALTZ,J. M., AND TERRY, R. D., Ultrastructural studies of multiple sclerosis, Lab. Invest., 20 (1969) 444-454.
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34 SuzuKi, K., PODUSLO,S. E., AND NORTON,W. T., Gangliosides in the myelin fraction of developing rats, Biochim. biophys. Acta (Amst.), 144 (1967) 376-381. 35 VANCE,n . E., AND SWEELEY,C. C., Quantitative determination of the neutral glycosyl ceramides in human blood, d. Lipid Res., 8 (1967) 621-630. 36 VIRCHOW,R., Ueber das ausgebreitete Vorkommen einer dem Nervenmark analogen Substanz in den Thierische Gewebe, Virchows Arch. path. Anat., 6 (1854) 562-572. 37 WALLACH,D. F. H., Isolation of plasma membranes of animal cells. In B. D. DAvis AND L. WARREN (Eds.), The Specificity of Cell Surfaces, Prentice-Hall, Englewood Cliffs, N.J., 1967, pp. 129-141. 38 WOLMAN,M., Myelin breakdown in vitro, Biochim. biophys. Acta (Amst.), 102 (1965) 261-268. 39 WORTHINGTON,C. R., ANDBLAUROCK,A. E., Low-angle X-ray diffraction patterns from a variety of myelinated nerves, Biochim. biophys. Acta (Amst.), 173 (1969) 427--435. 40 WRIGGLESWORTH,J. M., AND PACKER, L., pH-Dependent conformational changes in submitochondrial particles, Arch. Biochem., 133 (1969) 194-197.