Centrifugation of brain microsomes in a density gradient

Experimenial

332

CENTRIFUGATION

Institute

of Anatomy

OF BRAIN

GRADIENT

V. HAYZON

and G. TOSCHI’

of Biochemistry,

Received

October

21,

MICROSOMES

DENSITY

and Institute

Cell Research

University

of Uppsala,

332~3-f6

(1960)

IN A

Sweden

28, 1959

D-L KIUC; .

a previous investigation on brain microsomes, the separation of KNA-rich particles from the membrane structures \vas attempteti 11~ various procedures [l, 7, 151. The most cfl’ective technique provetf to be the selective removal of the membranes by nonionic detergents, but this can obviously involve extensive changes in size and composition of the particles. Stepwise centrifugation in isotonic sucrose solutions was of limitcci use and suggcsteti that the particles could be concentrated 1,~ utilizing differences in density This \\-as also suggesteti 1)~ the results bet\vern particles anti membranes. obtained with the anal$cal centrifuge anti 1)~ the fact that similar microsomal particles from other tissues show a particularly high tlcnsity [ 11, 121. Therefore in the present stuctg we have applied various centrifugation procedures, all based upon seciimcntation in a gratiient of increasing density, as previously tlescrihctl hy other workers [3, 5, 8, $11.

MATERIAL

AND

METHODS

Because the principal purpose of this study was the fractionation of a rather complex mixture, such as the whole microsomal fraction, into characteristic subfractions suitable for biochemical and morphological examination, true “equilibrium centrifugation” was not performed, except in particular experiments which were carried out using previously purified, homogeneous fractions. Instead, an attempt was made to obtain a practical and evident separation of the different microsomal structures in well defined zones of the gradient. The essential elements of the centrifugation procedures adopted are reported here; details are given in the sections on the various experiments. All operations were performed at 2”-4°C. The starting material submitted for fractionation was (a) standard microsomes, prepared by the method previously described [15] and carefully resuspended in a 1 Permanent address: Istituto Superiorc tli SanitB, Home (Italy). Experimental

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suitable sucrose solution. (b) whole “postmitochondrial supernatant” (PMS) obtained by centrifuging the rat brain homogenate (1 volume in 3 volumes of 0.32 M sucrose) at 20,000 x g for 15 min. This latter preparation is preferable for the following reasons: it gives a more complete picture of protein and RNA distribution in cytoplasm, it eliminates clumping of the microsomal structures packed by high speed centrifuging and it avoids the removal of soluble constituents which can be important for preserving them. Density gradients were prepared by layering sucrose solutions of different concentrations in the centrifuge tube. The tubes were then allowed to stand 20 to 26 hours in the cold before the run. The initial boundaries between the layers were marked on the wall of the tube and used as reference points at the end of the run. Both pH and ionic composition were kept constant in all the solutions at levels which, according to our experience and to general knowledge about microsomal particles [ 1, 4, 13, 14, 161, assured maximum stability. Thus 0.001 M MgCl, and K,HPO, (pH about 6.8) were present in all the solutions used-this solution will be referred to as “P-Mg buffer” throughout this paper. In some instances concentrations of 0.0005 JZ were also used without appreciable differences. As a rule, by adding 2 M sucrose, the starting preparations were brought up to a sucrose concentration (0.9 &I) such that they would not form a steep boundary when layered above the gradient. This was also avoided by gentle stirring of this boundary. The presence of a sharp demarcation between preparation and gradient can give rise to artifacts. One ml samples were layered above gradients having a volume of 4 to 4.5 ml. Volumes and characteristics of the adopted gradients are illustrated in Fig. 1. Centrifugations were performed in the swinging, horizontal rotor SW-39 of the preparative Spinco centrifuge Mod. L. Due to limits in the resistance of the available rotors, some of the experiments were run at 39,000 rpm (125,000 xg) and some at 31,000 rpm. Although a better separation was possibly obtained at the higher speed, essentially similar results were observed at the lower one. The time of centrifugation for effective separation was 3 hours, a period in agreement with the reported data on equilibrium centrifugation of subcellular fractions [5, 91 and virus particles [3]. Examination of the tubes at the end of the run was made by inspection against a black background with a lateral light, and by the simple optical arrangement reported by Anderson 121. The zones chosen for sampling were marked on the tube wall with waterfast ink and the tube was cut with a suitable apparatus such as reported b\ Hebb and \Vhittaker [Xl. The fractions obtained were examined routinely for their protein and RNA content by methods previously reported [ 151. Samples for morphological examination with the electron microscope were immediately fixed by adding one volume of 2 per cent 0~0, in buffered sucrose. After 2 hours the samples were diluted to 12 ml with the P-Mg buffer and centrifuged 2 hours at 105,000 xg. The resulting pellets were then rinsed, dehydrated and infiltrated with methacrylate monomer in an “automatic technician” [61, which changes the fluids continuously and thus gives a minimum disintegration of the loosely packed material. Ultrathin sections were examined in a RCA E!VfU-3B electron microscope. More details about the preparation technique were reported previously [l, 71. The micrographs presented here are selected to represent the mean of several sectioning turns from different parts of the pellets and of several experiments of the same type as indicated in Fig. 1. Bzperimenfd

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V. Hanzon and G. Toschi

334

EXPERIMENT

AND

RESULTS

Fig. 1 illustrates the different experiments carried out with various density gradients. The original boundaries are indicated by the lines on the sides of the tubes. The clear and shaded areas represent the visible zones observed at the end of the centrifugation. The fractions obtained by cutting are indicated by arrows. Corresponding to each fraction, the respective concentration of RNA (on a protein basis) is reported. The range of values observed in the different experiments is also given. Fig. 1 CI illustrates a series of experiments with the “postmitochondrial supernatant” PXIS, run on the gradient indicated, under the conditions reported above. From top to bottom, the following zones are observed at the end of the run: the starting zone is translucent and pinkcoloured, the hemoglobin apparently remains here with the soluble, non-sedimentable constituents (a thin crust of light lipid-rich material is often observed.) Then follows a wide zone of opalescent and almost colourlcss material, without any sharp upper or lower front, extending down to the original boundary 1.2-1.5 M. For sampling, as shown by the arrows, the translucent zone was taken as fraction A, while the opalescent zone was divided into t\vo halves, B and C. Finally, the completely translucent and colourless zone belo~v C was taken as fraction D. The analyses show that the RNA concentration in the top fraction A is remarkably constant around a value of 10 pg/mg protein. This cytoplasmic RNA. RNA concentration probably represents the “soluble” increases downwards in the two halves of the opalescent zone, B and C, \vhere it wrresponds to the values previously observed in microscomal preparations respectively less and more rich in particles [15]. Fraction I), from the boundary 1.2-l .5 to 1.5-l .8 M sucrose, shows values corresponding examination (Fig. 2) with those of purified particles. The morphological sho\vs a low content of particles in fraction B, more particles in fraction C and a great preponderance of particles in fraction D. Particles attached to membranes were not frequently observed, on the contrary most of them are grouped in clusters, in which they sometimes seem to be kept together by sparse, structureless material. In several of the experiments with centrifuRation in density gradients a particular membrane structure was observed, most remarkably in fraction B (arrow in Fig. 2). It consists of a membrane delimited body filled with a characteristic, homogenous and very opaque material. Often the membranes are arranged as two concentric rings in the sections, suggesting a double walled vesicular structure with the dense material between the two walls. Experimental

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Fractionation Gradient type

Number of experiments

sucrose molarity

335

of brain microsomes Fractions sampled

RNA concentration (pg/mg of protein) 722-

4

3

Yield (%)

11

(16-20)

30

(18-30)

76-128

(31-36)

181-266

(12-15)

S-

13

26-

49

226-335 361-410

29-

46

27-

71

131-134

2

210-367

0.32

d

2

1

L--.-

A

lo-

13

0

45-

66

C

131-240

310-393

4 1.5

U-

Pellet

6-

11

31-

44

240-306

Fig. l.-Scheme of the experiments. Fig. l.-The figure and the table illustrate the different experiments carried out with various density gradients. The postmitochondrial supernatant (PMS) was used in all the experiments, except in lc, where microsomes resuspended in 0.9 JII sucrose were used. Except in experiments 1 d where all the layers were prepared at the same time one hour before the run, 0.5 or 1 ml of the starting preparation was layered just before the run on top of the gradient prepared 20 to 26 hours before. Centrifugations were performed in the Spinco model L centrifuge, horizontal rotor SW 39, at 39,000 rpm for 3 hours. Fractions were taken by cutting the centrifuge tube. The original boundaries are indicated on the side of the tube. Shaded and clear areas represent the visible zones observed at the end of the run. The fractions isolated by cutting are indicated by arrows. In correspondence with each fraction the respective concentrations of RNA (on protein basis) are reported. The range of the values observed in the different experiments is also given. Experimental

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Fig. ‘L.--Electron micrographs of fraction 13, C and I) from centrifugation of “postmitochondrial supernatant” of brain homogenate in density gradient corresponding to Fig. In. Membranes decrease and particles increase in amount from R to D. Arrows point to the characteristic, membrane bounded, opaque structures described in the text. x 100,000. Expperimentnl

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of brain microsomes

Fig. 3.--Electron micrographs of fraction B, C and D from centrifugation of “postmitochondrial supernatant” of brain homogenate in density gradient corresponding to Fig. 1 b. More particles and less membranes in the lower fractions with higher density. Y 100,000. Experimental

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Fig. 1 b shows the results obtained by slightly modifying the gradient used in 1 a, thus giving a better separation of the particles from the mcmhranes. A higher RNA concentration is realized in fraction 11, as comparetl to 1 Q, and the electron micrographs show a high amount of particles although some membranes also occur (Fig. 3). Fig. 1 c gives the results obtained by centrifuging a standard preparation of microsomes (resuspended in 0.9 31 sucrose) on the same gradient as in 1~1. Proceeding from top to bottom, the starting zone contains some light, opalescent and faintly colouretl material (fraction A) followetl alternatel\ by two dense and two less dense zones, \\-hich end at the lwundary 1.2-l .5 .\I, as indicated in the drawing. The fractions sampled, as indicated 1)~ arro\vs, show a definite increase in RNA concentrations in fraction C anti 11 xvith a maximum in the latter, ahead of the opalescence. As to the morpholog\ (Fig. 4) fraction A and H sho\vs mainly membranes. In fraction C many particles appear and fraction 11 contains preclominantly particles with only a fe\v membranes. Thus, the same essential pattern is obtained with the postmitochondrial supernantant as with microsomes, hut this latter material appears less suitable for regular separation of memhrants and particles hy means of the gradient. Fig. 1 d illustrates cxpcriments with a simple, two-step gradient as used hy Hebb ant1 U’hittaker [a]. In this case the two sucrose solutions and the postmitochondrial supernatant (0.32 111 sucrose) \I\-ere layered at the same time, allowed to stand 1 hour, then centrifuged for 2 hours. After ccntrifugation, thick layers of densely packed material were obscrrcd in the zones of They were apparently artifacts due to the sutlticn the two steep houndaries. changes of density and of osmotic pressure with consequent packing OT the material at the houndaries. Going from top to hottom, a translucent anti coloured zone is observed, followed 1)~ a densely packed zone and a \vitic opalescent area ending in similarly dense zone A\-hich corresl~onds to the hounclary 1-2 M sucrose. The fractions, sampled according to thca arroxvs, show the usual low concentration of RN.1 in fraction A, xvhilc fraction B sho\vs the average values observed with the standard microsome’s, hut fracincrease of RSA1. tion C, including the lower packed zone, sho~2 a definite These values arc even higher in fraction I), the translucent colourless zont’ hclo\\- C. The electron micrographs (not represented here) show a high conFig. 4.-Electron micrographs of fraction A to D from ccntrifugation of a standard preparation of brain microsomes in density gradient as described in fig. Ic. In A and B mainly membranes, in C and D increasing amount of particles and less membranes. In C some particles are attached to membranes. x 100,000. Experimenful

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Experimentnl

339

Cell Resenrch 21

V. Hanzon and G. Toschi tent of membranes with few particles in H, zones of varying composition in C and mostly particles in D. On the basis of the results obtained from the gradients reported above, a gradient similar to 1 (I was used for preparative purposes, with the aim of obtaining highly concentrated, purified particles in a small volume. Centrifugation in a density gradient is actually the simplest and quickest procedure; it minimizes manipulation and undesirable changes of the medium anti avoids treatments with agents affecting the properties of the particles. A gradient such as illustrated in Fig. 1 e was prepared in the 13 ml tubes of the angle rotor n. 40. Four ml of the postmitochondrial supernatant mere laycrcd on top and run at 40,000 rpm for 3 hours. ,% thin crust and a top coloured zone corresponding to the starting zone were observed. Then an opalescent homogeneous zone extended downwards to the boundary 1.2-l .5 M, followccl by a colourless translucent zone. .At the bottom a small, thin, translucent, gelatinous and slightly pink-yellowish pellet was observed. RX.4 concentration in fractions A, B and in the pellet were of the same order as found in fractions A, B and D of the similar gradient 1 or). Electron micrographs (Fig. 5) show the presence predominantly of particles in the pellet, sometimes together with some remnants of mitochondrial membranes, which apparently have a higher density than mirrosomal membranes. The same “preparative” gradient was used for purification of particles in preparations treated with a non-ionic detergent. It is kno\vn from previous investigations [7, 10, 1S] that this treatment dissolves most of the microsomal membranes but remnants of these arc still found \vith the particles \vhcn they are spun down in isotonic sucrose. In the present experiments Lubrol W was added to the PhlS 30 min before the run on the preparative gradient rcported above. The particles recovered in the pellet showed the highest degree (Fig. 6). The of purity so far obtained, as indicated by the micrographs highest RNA concentration (350-JO0 /cg/mg protein) was also observed in this pellet. Apparently the remnants of membranes are retained by the upper zones of the gradient. Centrifiqcltion crppro0ching eqrlilibriulrz was performed on purified particles obtained using the preparative gradient described above, 1 e. This material, resuspended in 1 ml of 1 M sucrose, was layered on top of a gradient formed by 5 layers (1.2-l .5-1.X-2-2.5 M sucrose) and was centrifuged for

centrifugation of “postmitochonFig. 5.--Electron micrograph of the pellet from “preparative” drial supernatant” in density gradient according to Fig. 1 c. Mainly particles are visible irregularly arranged in clusters. x 100,000.

Esperimental Cell

Hesearch

21

Fracfionafion

22 - 60173’53

of brain microsomes

Experimental

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V. Hanzon and G. Toschi

I;ig. 6.--Electron postmitochondrial sharply delimited x 100,000. ExDerimental

micrograph of the pellet from “preparative” centrifugation of Lubrol-treated supernatant in density gradient as in Fig. le. Only particles are visible, when they are single (arrows) but mainly arranged in clusters of different sizes.

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Fractionation

of brain microsomes

Pig. 7.--Ultracentrifugal analysis of a microsomal preparation rich in particles. Medium: 0.22 AI sucrose in 0.05 AII phosphate buffer pH 7.6 and 0.01 M Mg Cl,. Run at 39,460 rpm, at 20”. Exposures after 4, 8 and 12 min. The main peak C has a sedimentation constant (S,,, approximated) of 67 S.

Fig. 8.--Ultracentrifugal analysis of purified particles which were prepared from microsomes by treatment with Lubrol W and centrifugation in the “preparative gradient” (see page 340 and Fig. 6). Medium: 0.05 :I1 phosphate buffer pH 7.6 and 0.01 M Rig Cl,. Run at 39,460 rpm, at 20”. Exposures after 4, 8 and 12 min. The main peak has a sedimentation constant (S,,, approximated) of 74 S.

7 hours. d slightly opalescent, but well defined zone was ohservcd which corresponded with the original layer of 1.8 ;14 sucrose. This is apparently the density which particles tend to reach at equilibrium in such a gradient. INV’ESTIGATION

WITH

THE

ANALYTICAL

CENTRIFUGE

These examinations have been performed on some microsomal preparations which xverc being used for density gradient centrifugation, as well as Experimental

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V. Hanzon and G. Toschi

on purified preparations of particles obtained by such a procedure. Various media with dif’ferent ionic compositions were tried. Neither the microsomal starting preparations nor the purified particle preparations, when suspended in a medium of the usual composition (0.001 or 0.0005 M hIgC1, and K,HPO,) and pH 6.8, did show a definite boundary with a sedimentation constant such as would be expected for this class of particles [13]. They apparently contained only a bulk of aggregated opalescent material xvhich quickly packed in the bottom of the cell. However, in contrast, the appearance of well defined boundaries with sedimentation constants in a range of 40 to 80 S was observed when the same preparations were suspended in a medium of both higher pH (7.6) and ionic strength (0.05 M phosphate bufier), the concentration of MgC1, being 0.001 dl or less The concomitant presence of components with (lilTwent (Figs. 7 and 8). sedimentation constants is also observed in these patterns, suggesting that both aggregation and splitting of the particles occur.

DISCUSSION

In the present \vork the ccntrifugation in density gradients of various types has allowed the separation of frac,tions characterized by difrerent amowlts of the biochemical and morphological constituents of brain microsomes. In agreement with the starting hypothesis, in all cases an evident increase ol the concentration of both RNA and particles from the upper to the lower zones of the gradients \vas shown. This separation was more regular \\-hen the l~ostmitochon~lrial sulwrnatant was used, with gradients of the continuous type. The membrane structures \vcrc more or less continuously distributed over a wide opalcxent zone from 0.9 JI to 1.5 M sucrose, those collecting in Ihc top zone being devoid of attached particles whereas vesicles \\-ith attached particles were frequent in the lo\vcr zones (Fig. 4 c). This indicates a tcndenc? to the separation of two kinds of membranes, in the denser and less clensc rcspcctivdy. On the other hand 1nos1 zones, “granular” and “agranular” are not clearly attached to of the particles, which collect in the denser ZOIIPS, membranes but they rather appear to form aggregates of’ various size. The particular membrane structures remarked in fraction H of the gradients 1 a, 0 and c cannot bc identified with knolvn cytoplasmic structures of neurons. They A\-cre occasionally observed in previous examinations of standard microsomes [i], but they were very rare. Thus a definite concentration of them in zones of low densit? can be obtained with the density gradients. E.rperimenfnl

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The fractionation of microsomes from amphibian eggs in similar gradients, performed by Ottesen and Weber [9], was similar in that it showed a continuous distribution of opalescent material over an extended zone in the upper part of the gradient. The present procedures have proved effective for a quick and simple preparation of purified microsomal particles, both from untreated and detergent-treated microsomal preparations. The yield of both RNA and particles from untreated material is however low, a great part of the RNA-rich particles still remaining within the membrane-rich fractions. It is suggested that a similar enrichment of particle-free membranes is possible. The results obtained by the examination with the analytical centrifuge point out that practically all the particles are in form of aggregates, employing the medium as in the present experiments (0.001 111K,HPO, and MgCl,). This confirms the electron microscopical observations, which show that the particles are grouped in clusters (especially obvious in Figs. 5 and 6). The apparent liberation of particles from the aggregates, induced by the medium with high pH and ionic strength, and associated with some splitting, is in agreement with a recent report of Petermann et al. on liver microsomes [13]. The sedimentation constants, about 70 S, observed by us on the particlcs prepared and examined in presence of Jig, agree fairly well with their size as shown by the electron micrographs and with their density as indicated by the centrifugation in density gradients. These combined characteristics are similar to those of liver microsomal particles, as reported by other authors [12, 131. SUMMARY

Microsomal preparations from rat brain were centrifuged in various density gradients obtained with different concentrations of sucrose. The different zones of the gradients were analyzed chemically as to their protein and R?;A content and examined with the electron microscope as to their content of particles and membrane structures. A satisfactory separation of the two components of the microsomal preparations was obtained. The membranes \vcre recovered in the upper zones with 101~ density, whereas the particles were obtained in high amount, with only a few membranes left, in the lower zones, with high density. The concentration of particlcs was fairly well correlated with the concentration of RNA in the different zones, thus giving further evidence of the association of RNA with the particle component of the microsome preparations. The centrifugation in density gradient represents a quick and simple method for the isolation of RNA-rich particles. Experimental

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We wish to express our thanks to Prof. A. Tiselius for his interest in this work. It has been aided by a grant from the Swedish Medical Research Council. Dr. iK. Marsden kindly revised the English. REFERENCES 1. 2. 3. 4. 5.

ALDERTSSON, P. A., HANZON, v. and TOSCIII, G., J. Cftrasfructure Research 2, 366 (1959). ALYDERSSON, N. G., Biochim. et Biophys. Acfu 25, 428 (1957). URAKKE, M. I<., Virology 6, 96 (1958). CHAO, F. C., Arch. Biochem. Biophys. 70, 426 (1957). DE Duve, C., BERTET, J. and BEAUFAY, H., Progr. in Biophys. and Biophys. Chem. 9, 32.5

(1959). 6. 7. 8. 9. 10. 11. 12. 13.

HANZON. V.. Science Too/s 6, 18 (1959). HANZON, \:.‘and TOSCHI, G.,.Expil. Cell Research 16, 256 (1959). HEBB, C. 0. and \\‘HITTAKER, V. I’., .I. Physiol. 142, 187 (1958). OTTESEN, M. and WEBER, R., Compt. rend. Lab. Carlsberg Skr. chim. 29, 417 (1955). PALADE, G. E. and SIEKEVITZ, P., .I. Biophys. Biochem. Cyfol. 2, 171 (1956). PETERM&N, 31. L., Texas Repfs. Biol. aied: 12, 921 (1954). PETEHMASN, hf. L. and HAMIC~ON, nl. G., J. Biol. Chem. 224, 725 (1959). PETERMANN, M. I,., HAMILTON, M. G., I~ALIS, 3%. E., SAMART, I<. and PECORA, P., in Micro-

somal Particles and Protein Synthesis, p. 70. Roberts R. B., Ed., Pergamon Press, J.ondon, 1958. 14. TISSIBRES, A. and WATSON, J. D., Knfure 182, 778 (1958). 15. TOSCHI, G., Expfl. Cell Research 16, 256 (1959). ,J., Biochim. ef Biophys. dcfa 30, 570 (195X). 16. T’so. P. 0. P., BONNER, J. and VINOGRAD.

Experimenfal

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