FREE-FLOW ELECTROPHORETIC SEPARATION

AND ELECTRICAL SURFACE PROPERTIES OF

SUBCELLULAR PARTICLES FROM GUINEA PIG BRAIN

K : J . RYAN, H . KALANT, and E . LLEWELLYN THOMAS

INTRODUCTION

Previous isolations of brain subcellular particleshave been achieved primarily by means of dif-ferential centrifugal techniques, which have beenreviewed by Whittaker (1) and De Robertis (2) .Kadota and Kanaseki (3) have combined centrifu-gation and diethylaminoethyl(DEAE)-Sephadextechniques to purify, synaptic vesicle fractions . Voset al . (4) and Sellinger and Borens (5) have usedthe zonal density gradient electrophoresis methodof Svensson (6) in the further fractionation ofcentrifugal preparations of brain subcellularparticles .

In the present work continuous free-flow electro-phoresis, as described by Hannig (7), has beenused to increase the purity of fractions of sub-cellular particles, to reduce the preparation time,and to allow electrokinetic interpretation of dataobtained during the separation steps. This paperdescribes the optimum electrophoretic separationconditions. for preparations of synaptosomes,mitochondria, and synaptic vesicles, as well as

From, the Department of Pharmacology and Institute of Biomedical Electronics, University ofToronto, Toronto, Canada

ABSTRACT

Continuous free-flow electrophoretic separation has been used to obtain relatively purepreparations' of synaptosomes and synaptic vesicles from crude fractions of guinea pig brainhomogenates . Measurements of the contents of protein, neuraminic acid, and bound,'acetyl-choline; the activities of succinic dehydrogenase, adenosine triphosphatase, choline acetyl-ase, and 5'-nucleotidase ; and the uptake of .14C-labeled choline and atetylcholine in thepresence and absence of hemicholinium, all confirm the electron microscope evidence thatthe electrophoretic preparations are at least as pure as those obtained by ultracentrifugalmethods. The electrophoretic mobility measurements have been Wed . to calculate zeta po-tentials and surface charge densities for these particles .

some morphological and biochemical studies ofthe isolated fractions ..

METHODS

Centrifugal Preparations

Four types of centrifugal preparation of guineapig cortex were used in this work. All four fractionsware isolated by the methods outlined by Whittaker(1) and are designated, using Whittaker's termi-nology, as : supernatant of crude homogenate, crudesynaptosomal-mitochondria) fraction (P2), purifiedsynaptic vesicle fraction (DI), and purified synapto-somal fraction (P2B) .

Electrophoretic Isolation

The apparatus used for the electrophoretic . sepa-rations was originally designed by Hannig and ismarketed as the Brinkmann FF-3 Continuous FreeFlowing Electrophoretic ' Separator (BrinkmannInstruments •Inc., Westbury, N.Y.) . The principle of

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operation may be briefly outlined as follows : thesample to be separated flows vertically downward ina rectangular chamber measuring 50 cm in length,12 cm in width, and 0.5 mm in thickness . Thevertical velocity of the sample is equal to that ofthe flowing chamber buffer . The individual particlesof the sample move horizontally with a velocitydetermined by the electric field applied across thechamber and the electrostatic characteristics of theparticles . The continuously injected sample dividesinto bands, each containing particles of equalmobility ; the bands are then isolated at the bottomof the separating chamber by a manifold of 92 tubes .

Distribution of Material in Output Fractions

The output fractions of the separator were ana-lyzed by nephelometry using a Photovolt 520Mfluorimeter (Photovolt Corporation, New York)in order to determine quickly the distribution ofparticulate matter in the array of collector tubes .

Electron Microscopy

Samples were studied and photographed in aZeiss EM9A electron microscope. For negative stainpreparations, 1-2% (w/v) phosphotungstic acid,pH 7.0, was used according to the methods of Horneand Whittaker (8) and Whittaker (9) . Positive stainfixation was done according to Farquhar and Palade(10) . Sections were cut on a Porter-Blum MT2microtome, and stained with 6% uranyl acetateand Reynold's lead citrate solutions .

Mobility MeasurementThe total deflections of the individual bands of

particles within the separating chamber were re-corded photographically. The lateral displacement ofparticles was made up of one component due to theelectrophoretic velocity of the particles and a secondcomponent due to the electro-osmotic velocity ofthe chamber buffer . To correct for the latter com-ponent a zero mobility marker (chamber buffercontaining 0 .4 M sucrose) was injected into the cham-ber. The resultant diffraction pattern, establishedat the interface between the marker containing0.4 M sucrose and the normal chamber buffer con-taining 0.32 M sucrose, could be observed visually .A fine thread was used to mark the position of theinterface for photographic recording . The angle ofdeflection of the bands due to the electrophoreticvelocity of the particles was measured as the differ-ence between the angles of total deflection of theindividual bands of material and the angle of de-flection of the marker .

The volume flow rate of the chamber buffer wasmeasured directly by collection of the output of theseparating chamber for a measured time interval .

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THE JOURNAL OF CELL BIOLOGY . VOLUME 49, 1971

The maximum linear flow rate of the chamberbuffer was calculated from the volume flow rate andthe geometry of the separating chamber . The chamberbuffer was assumed to exhibit laminar flow, with aparabolic velocity profile across the narrowest di-mension of the chamber.

The electric field within the chamber was de-termined directly by measuring the voltage dropbetween two test probes of measured separation .The mobility of a given band of particles was calcu-lated using the formula :

u = Vm . x [tan Bb - tan 0m],s

where u = electrophoretic mobility, Vmax = maxi-mum vertical velocity of the buffer, E = electricfield within the chamber, Bb is the angle of deflectionof the band of particles, and Bm is the angle of de-flection of the zero mobility marker .

14C-Label Studies

Choline chloride (methyl- 14C), SA 54 mCi/mmole, and acetyl- 1- 14C-choline chloride, SA 9 .2mCi/mmole, were used as markers for mitochondriaand synaptosomes . For a validation of the use ofthese markers, see the uptake experiments of March-banks (11, 12) and Burton (13) . P2 crude prepa-rations of mitochondria and synaptosomes wereincubated in the presence of 5 µCi ofcholine chloride(1 yCi/ml) or 10 sCi of acetylcholine chloride (2,uCi/ml) in a medium described by Marchbanks (11) .In the acetylcholine experiments, eserine was in-cluded in the medium (0 .1 mg/ml), and also non-labeled acetylcholine chloride (50 mm) . The P2fraction was divided into two samples of equal vol-ume, one of which was incubated in the presence ofeither choline- 14C or acetylcholine- 14C, and theother of which was incubated in the presence of thesame labeled compound and hemicholinium No. 3(100 µM) . After a preincubation of 20 min at 25 °C,the labeled compound was added to each sampleand the incubation was continued for 30 min. Atthe end of the 50-min period the samples werecentrifuged at 17,500 g for 30 min and resuspendedin chamber buffer. Electrophoretic separation wascarried out for 45 min . The output fractions of theseparator were then analysed by liquid scintillationcounting techniques . 1 ml of each fraction was addedto 10 ml of Bray's (14) solution and counted by thechannels ratio method in a Nuclear-Chicago LiquidScintillation Counter Model 720 (Nuclear-Chicago,Des Plaines, Ill .) .

Biochemical Assays

Neuraminic acid concentrations were measuredby the procedure of Warren (15) with the following

modification . Each electrophoretic fraction wascentrifuged to a pellet (18,000 g), resuspended in 1 .0I111 of 0 .1 N H2SO4, and incubated for 1 .0 hr at 80 •C .The hydrolyzed sample was placed in a narrowdialysis sac and dialyzed against 10 ml of twice-distilled water at room temperature, with continuousshaking, for 24 hr . A blank of H2SO4 without tissuewas carried through the same procedure. The 10ml of outer phase was evaporated under nitrogen to avolume of 0.3 ml . Subsequent steps followed Warren'smethod. Graphical techniques were used to deter-mine the N-acetylneuraminic acid levels after spec-trophotometric scanning from 420 to 600 mé . Purestandards were used for comparison in each experi-ment.Na,K-ATPase and ouabain-resistant ATPase

activities were determined by the method of Postand Sen (16) . Succinic dehydrogenase activitywas measured by means of the Warburg respirome-ter technique, with the method outlined by Umbreitet al. (17) .

Measurement of 5'-nucleotidase activity wasdone by the method of Heppel and Hilmoe (18) .Choline acetylase activity was measured by themethod of Berry and Whittaker (19) . Bound acetyl-choline content in fractions of the electrophoresisoutput, as well as acetylcholine produced in thereaction mixtures for choline acetylase activity,were measured by the spectrophotometric method ofHestrin (20), initially validated against the frogheart bioassay method of Zapata and Eyzaguirre(21) . For this purpose, samples were adjusted topH 4-4.5 by addition of dilute (0.2 N) HCl, heatedat 100 •C for 10 min, cooled, and centrifuged for 10min at 8000 g. The supernatant was used for analysis .Protein was determined by the method of Lowryet al . (22) .

RESULTS

Optimum Separating Conditions

Preliminary experiments were undertaken witha crude preparation (supernatant of 1000 g X 11min) of guinea pig cortex homogenized in 0 .32 Msucrose (10% w/v) at pH 7 .0. The heterogeneousmixture was fractionated in the Brinkmann FF-3,using five different buffers and the following rangeof operating conditions : chamber voltage 550-1200 v, chamber current 50-150 ma, pH 4 .5-8 .6,buffer flow 0 .04-0.117 ml/sec, and chamber con-ductivity 4.1 X 10-`-6.64 X 10-4ohm1-cm1 .

Fig. I indicates the conditions which producedoptimum separation as judged by turbiditymeasurements performed on the electrophoreticfractions of a crude mitochondrial-synaptosomalpreparation. The nephelometric measurements

35 45 55TUBE NO .

65 75

FIGURE I Turbidity profile of the electrophoretic out-put fractions of a crude mitochondrial-synaptosomalpreparation . The injected material was the P2 fractionfrom guinea pig cortex prepared according to Whittaker(1) . The separation conditions were : 1900 v, 55 ma,6 •C, electrode buffer pump setting 50, and dosing pumpsetting 10 .1 . Michaelis-Veronal buffer of pH 7 .15 andconductivity 4 .1 X 10-4 ohm1cm1 was used for thechamber buffer . The broad peak appears to consist oftwo incompletely resolved peaks .

indicated two distinct peaks and a wide separationof material . When a centrifugal pure preparationof synaptosomes (P 2B) was run under the sameconditions as in Fig . 1, the main distribution oc-curred around tube 68, the location of the sidepeak in Fig . 1 . This provided preliminary evidencethat synaptosomes could be isolated from a verycrude preparation .

Electron Microscopy

The synaptosomal-mitochondrial preparation P 2and the synaptic vesicle preparation D 1 wereindependently subfractionated by electrophoresisunder the conditions given in Fig . 1 . The sub-fractions were then studied by electron micros-copy .

Fig. 2 shows a negative stain preparation offraction D1 before electrophoresis . This fractionresembles closely the synaptic vesicle preparationsdescribed by Whittaker (1) and De Robertis (2) .Note the white-cored and hollow-cored vesicles,broken vesicles, vesicles on edge, and contamina-tion by stringlike figures and larger particles .

After electrophoresis of D 1 a fraction composedprimarily of white-cored vesicles (Fig . 3) was ob-tained; the major contaminant materials were

RYAN, KALANT, AND THOMAS Subcellular Particles from Guinea Pig Bran 237

FIGURE 2 Electron micrograph of the synaptic vesicle fraction, D1, prepared from guinea pig cortexaccording to Whittaker (1) . The negative stain methods of Horne and Whittaker (8) were used . Arrowsindicate hollow vesicles (h), white-cored or "solid" vesicles (s), and vesicles in profile (p) . Bar = lµ.X 51,000.

FIGURE 3. Electron micrograph of one electrophoretic subfraction of the same fraction, D1, as is shown

in Fig . 2 . The conditions used for separation were the same as in Fig . 1 . Note the increase in the relativenumber of white-cored vesicles, clumps of which are indicated by arrows . Negative stain was used .Bar

lµ . X 51,000 .

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THE JOURNAL OF CELL BIOLOGY • VOLUME 49, 1971

FIGURE 4 Electron micrograph of a synaptosomal-mitochondrial preparation, P2, from guinea pig cortexprepared according to the method of Whittaker (1) and observed using the positive stain proceduresof Farquhar and Palade (10) . Three main types of particles were observed : synaptosomes, myelin, andmitochondria . Bar = 1µ . X 16,000 .

FIGURE 5 Electron micrograph of a synaptosomal fraction purified by electrophoresis from the con-trol material, P2, shown in Fig. 4 . Bar = lµ . X 16,000 .

RYAN, KAI,ANT, AND Tnoan s Subcellular Particles from Guinea Pig Brain

239

FIGURE 6 Electron micrograph of a mitochondrial fraction purified by electrophoresis from the controlmaterial, P2, shown in Fig. 4 . Bar = 1µ . X 16,000 .

found in an adjacent fraction in the outputspectrum .

Fraction P 2 is represented by Fig. 4 whichcontains three main types of particles : Into •chondria, synaptosomes, and myelin. After con-tinuous electrophoresis P 2 was resolved into twodistributions . The band of particles showing thehighest negative charge (i .e . migrating to thepositive electrode side of the main protein peak)was made up primarily of synaptosomes, as canbe seen in Fig . 5. This band corresponded to theside peak at tube 68 in Fig . 1 . The second bandwas made up of two poorly separated distributions :mitochondria (Fig . 6) which were displaced leastand collected on the negative electrode side of thelowest mobility band, and unseparated materialwhich constituted the major portion of the lowmobility band . In Fig . 1, tube 53 corresponds tothe center of the mitochondrial distribution .

Mobility Measurements

An example of the bands produced by the elec-trophoretic subfractionation of the crude mito-chondrial-synaptosomal preparation P2 is given inFig . 7 . Fig. 8 represents the angle of deflection dueto the electro-osmotic velocity of the buffer . The

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THE JOURNAL OF CELL BIOLOGY . VOLUME 49, 1971

required angles of deflection were measured in7 X 10 inch prints made from photographs takenduring repeated separations of P 2 and Dl fractions.The mobilities of synaptic vesicles, synaptosomes,and mitochondria were calculated from themeasured deflections, the observed chamber bufferflow rates, and the determined field strengthswithin the separating chamber (Table I) . Theorder of increasing magnitude of mobility for thethree particles agrees with that reported by Voset al. (4) . However, the individual magnitudesappear to be about twice as large .

Table I also includes the zeta potentials of thethree subscellular particles, calculated from themobility data and buffer characteristics by ap-plication of Henry's (23) equation according tothe theory reviewed by Overbeek and Lijklema(24) . The method and tabulated solutions of Loeband Wiersema (25) were used to determine thesurface charge densities of the three particles. Fulldetails of these calculations have been providedelsewhere (26) .

Radioactive Labeling

Fig. 9 illustrates the distribution of acetylcholine-I4C in the electrophoretic output fractions after

FIGURE 7 Bands produced by the electrophoretic subfractionation of the mitochondrial-synaptosomalpreparation P2 . The bands were photographed through the separating chamber face plate. The verticalline on the right represents the "zero mobility" reference line . The oblique line (A) farthest from thereference line is the synaptosomal band . The fraction corresponding to this band is shown in Fig . 5. Thelower mobility band (B) corresponds to the mitochondrial fraction and impurities shown in Fig . 6 .

FIGURE 8 The angle of deflection due to the electro-osmotic velocity of the buffer . Chamber buffer con-taining 0.4 M sucrose was injected into the normal chamber buffer which contained 0 .82 M sucrose . Awhite thread was superimposed on the resultant diffraction pattern to allow a photographic record ofthe angle of deflection caused by the motion of the chamber buffer under the influence of the appliedelectric field and other conditions used in the separation procedure .

TABLE I

Electrical Characteristics of Synaptic Vesicles, Synaptosomes, and Mitochondria

* Values shown indicate mean ±SE .

incubation of the P2 pellet in the presence of eserine best separation of a series of seven which yielded(0.1 mg /ml), unlabeled acetylcholine (50 mm), and essentially the same information .acetylcholine14C, and subsequent electrophoretic

The smaller peaks in the curves for disintegra-separation for 45 min. The curve represents the tions per minute indicate the position of the

RYAN, KALAN;, AND THOMAS Subcellular Particles from Guinea Pig Brain

241

ParticleNo . of

repetitions Mobility Zeta potentialSurface charge

density

cm2 X 104/volt-sec me µcool/cm2

Synaptic vesicles 4 2 .08 f 0 .05* -69 .2 f 2 .0 -1 .35 f 0.06Synaptosomes 2 3 .63 f 0 .04 -90 .3 f 0 .6 -1 .87 f 0 .01Mitochondria 6 2 .71 t 0 .23 -67 .5 t 3 .5 -1 .09 f 0 .09

3 .6-Q

2 .8 -

A

f""2 .0 -

,

i

I

1

k'k ~%

a .4-

40II

1.2 -

44

48

52

56TUBE NO .

- 72

- 56L

0- 40 2

FIGURE 9 Distribution of acetylcholine- 14C in theelectrophoretic output fractions of a P2 preparationseparated under the conditions shown in Fig . 1 . TheP2 preparation was preincubated for 20 min at 25 •C ina medium described by Marchbanks (11) . 50 ram un-labeled acetylcholine, 0 .1 mg/ml eserine, and 10 p..Ci ofacetylcholine- 14C were added and the incubation wascontinued for 30 min . The material was then spun down,resuspended in chamber buffer, and separated by free-flowing electrophoresis for 45 min. Relative positions ofthe chamber electrodes are indicated by + and - . Theoutput fractions were analyzed by liquid scintillationcounting techniques. A represents the disintegrationsper minute of the collected output fractions ; D repre-sents the effect of the addition of 100 / .AM hemicholiniumNo. 3 to the incubation medium, on' the disintegrationsper minute . B represents the specific activities of thesamples, and C the effect of 100 th hemicholinium No . 3on the specific activities . Specific activities could not becalculated over the full range because protein contentwas too low to measure accurately beyond tube 48 or49 .

synaptosomal band (positive electrode side of theprotein peak) ; the larger peaks indicate the positionof bulk unseparated material. It can be seen thatthe specific activity curves increase substantiallyin the vicinity of the synaptosome peak . In everyexperiment hemicholinium reduced the uptake ofthe labeled acetylcholine into the material makingup the two peak regions .

Fig. 10 shows the distribution of choline- 14Ctaken up by the major constituents of the P2fraction. The specific activity curves indicate twopeaks: the synaptosomal peak to the positiveelectrode side of the protein peak (tube 56) andthe mitochondrial peak to the negative electrodeedge of the main protein peak (tube 47) . Again,hemicholinium was found to reduce the uptake ofcholine in each experiment . Fig. 10 represents the

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THE JOURNAL OF CELL BIOLOGY é VOLUME 49, 1971

best separation in a series of five with similarresults .

Neuraminic Acid

Table II indicates the distribution of N-acetyl-neuraminic acid (NANA) in the, electrophoreticsubfractions of the synaptosomal-mitochondrialpreparation (P2) . The results are taken from one oftwo experiments which provided identical in-formation concerning the relative distribution ofmaterial . The highest specific concentration andrelative specific concentration of NANA occurredin the synaptosomal band, which in,' all the"marker" studies was 'consistently the band withhighest mobility . The values corresponding to tube48 in Table II are comparable to those obtainedby De Robertis (2), Scllinger,and, Borens (5), andLapetina et al. (27) for ultracentrifugal synaptoso-mal preparations .

ATPase Activity

Fig. 11 represents the distribution of ouabain-resistant ATPase and Na,K-ATPase in theelectrophoretic subfractions of the (P 2) mito-

TUBE NO .

FIGURE 10 Distribution of choline- 14C in the electro-phoretic output fractions of a P2 preparation separatedunder the conditions shown in Fig . 1 . The procedurewas similar to that described in Fig . 9 except thateserine and "cold" choline were not added to the incuba-tion medium. A represents the disintegrations perminute of the electrophoretic output fractions ; D repre-sents the effect of 100 AM hemicholinium No . 3 on thedisintegrations per minute curve . B represents thespecific activities of the samples, and C the effect of 1001M hemicholinhim No . 3 on the specific activities . Elec-trode positions indicated as' in Fig . 9.

TABLE II

Distribution of N-Acetylneuraminic Acid in theElectrophoretic Subfractions of the Mitochondrial-

Synaptosomal Preparation (P2)

* Relative specific concentration indicates theamount of NANA in a given tube, as per cent oftotal NANA in the whole output array, divided byprotein content of the same tube expressed as percent of total protein in the whole output array .

1 Tube in which protein peak occurred .

chondrial-synaptosomal preparation . Again thedistribution is consistent with the location of themitochondria to the negative electrode side of the

main protein peak, as evidenced by high ouabain-resistant ATPase activity. The high Na,K-ATPaseactivity to the positive electrode side of the mainprotein peak is indicative of the location of

synaptosomes . Comparison of the levels of ATPase

activity in tubes 43 and 48 with the results ofWhittaker (28) and Hosie (29) suggest that theseparation of synaptosomes and mitochondria isequivalent to that obtained by ultracentrifugalmethods.

Succinic Dehydrogenase

The cumulative 140 min oxygen uptake of theelectrophoretic subfractions of the (P 2) mito-chondrial-synaptosomal preparation is given inFig. 12. The specific activity curve suggests thatmitochondria are located to the negative electrodeside of the main protein peak .

Choline Acetylase Activity and BoundAcétylcholine Content

Preliminary experiments, carried out before theelectrophoretic separations described above, hadconfirmed Whittaker's observations on the dis-tribution of bound acetylcholine in ultracentrifugalsubfractions of the crude mitochondrial-synaptoso-mal fraction of guinea pig brain homogenates (1) .

The maximum relative specific activities found foracetylcholine (percent of total brain acetylcholinecontained in a given fraction, divided by per cent

of total weight) were 15 .95 for the upper layer offraction D, (synaptic vesicles) and 4 .25 for fractionB (synaptosomes) .

0 0.6

a

1 .0

0.2

49

FIGURE 11 Distribution of ouabain-resistant ATPaseactivity (Ouabain) and Na,K-ATPase activity (Na,K)in the electrophoretic subfractions of the (P2) mitochon-drial-synaptosomal preparation. ATPase activity wasmeasured by the method of Post and Sen (16) .

53 57TUBE NO .

61 65

2.0

i0.8 WY

o.a

0 .4 Zw2-

ô

FIGURE 12 Distribution of succinic dehydrogenaseactivity in the electrophoretic subfractions of the (P2)mitochondrial-synaptosomal preparation . Cumulativeoxygen uptake at 140 min was measured by means ofthe Warburg apparatus as outlined by Umbreit et al .(17) . The tubes above No. 55 showed too little activityand too little protein for accurate measurement.

RYAN, KALANT, AND THOMAS Subcellular Particles from Guinea Pig Brain

243

441 1 .23 6.40 0.44345 1 .42 9 .60 0 .66346 2 .26 15 .8 1 .0947 2 .50 24 .6 1 .63 -48 1 .48 31 .8 2 .2749 0.474 29 .7 2 .0150 0.190 23 .8 1 .41

Tube No.Concentra- Specific Relative specific

tion concentration concentration

µg µg/mg protein % NA NA/% protein

ZwF0a0

wa2 .2~â

0-1

0â rc 2 .0

E W 1 .80f

0 °1 .4-

.0 .8

w y

w0 N5 a Lo -J }Y Fw OJo .eV Q< w0.6-0 ZZ Jo= =0.4-w U

0 .2-

---0

-50

FIGURE 13 Distribution of bound acetylcholine (A)and of choline acetylase activity (O) in the electro-phoretic subtractions of the (P2) mitochondrial-synap-tosomal preparation from guinea pig brain . Values areexpressed in relation to the protein content of each sub-fraction, and are superimposed on the curve showingnephelometric estimation (0) of distribution of totalparticulate material in the same experiment .

The distributions of choline acetylase activityand of bound acetylcholine content among theelectrophoretic output fractions of the (P 2) mito-chondrial-synaptosomal preparation are shown inFig. 13. About 75% of the identified cholineacetylase activity and of the bound acetylcholinemeasured in the various fractions was containedin the synaptosomal peak . In the experimentillustrated, the total recoveries for all fractions, inrelation to the initial amounts in the equivalentvolume of crude brain homogenate, were 44% forcholine acetylase activity and 49% for boundacetylcholine . The loss presumably reflects acombination of losses during the initial centrifugalpreparation of the crude mitochondrial-synaptoso-mal fraction, losses by inactivation during elec-trophoresis, and distribution of small unmeasurableamounts among other tubes in the output array .The peak concentration of bound acetylcholine(21 .5 Etg/mg protein, in tube 64 of Fig. 13) com-pares very favorably with that found in Whittaker'sfraction B in the preliminary experiments, whenallowance is made for the conversion of proteincontent to equivalent fresh weight .

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THE JOURNAL OF CELL BIOLOGY - VOLUME 49, 1971

°~ -100

5'-Nucleotidase Activity

Measurement of 5'-nucleotidase activity did notprove very satisfactory as an index of biochemicalspecificity of the fractions . Preliminary investiga-tions on rat brain homogenates showed an activityof 9.6 µmoles hydrolyzed/hr per ml of homogenate .Guinea pig brain showed a considerably loweractivity, amounting to about 4.5 µmoles/ml perhr. In the experiment corresponding to Fig. 13,measurable activity was found only in tubes 62-66, with a peak concentration in tube 63 . Thetotal recovery in these tubes amounted to only18.5% of the original input, with 5.6%0 in tube 63 .The reasons for the poor recovery have not yetbeen ascertained .

DISCUSSION

By means of continuous free-flow electrophoresis,preparations of synaptosomes have been obtainedwhich compare favorably with fractions isolatedby ultracentrifugal techniques. Examination byelectron microscopy, and measurements of severalindependent biochemical markers, indicate thatthe preparation obtained electrophoretically is atleast as pure as the best centrifugal preparationsdescribed in the literature . A significant advantageof the electrophoretic method is that the total prep-aration time is at least 3 hr shorter than that re-quired for the ultracentrifugal methods .

On the other hand, this electrophoretic pro-cedure has distinct limitations . Because of thelow volume of sample which can be isolated perhour, and the intrinsically short period of viabilityof certain biological materials such as synapticvesicles, purity of the preparations is obtained atthe expense of the yield . For preparations ofgreater stability, this limitation would not apply .There is little probability that the yield can be im-proved substantially, except by increase in thescale of the electrophoretic apparatus . The limit-ing feature is the ratio of linear flow rate of cham-ber buffer to rate of lateral displacement of par-ticles in the electrical field . Optimization of thisratio limits the rate of sample injection relativeto the buffer flow rate, while the rate of electro-phoretic migration is limited by the optimalcomposition of the buffer as explained below,and by the capacity of the refrigerating systemwhich cools the electrophoretic chamber . Themethod is therefore of major value for rapidpreparation of small samples .

A second constraint on the method is the prob-

lem of compatibility of the chamber buffer withthe biological particles undergoing separation,and with the electrical current requirements ofthe separator. The buffer must not only be ofsuitable tonicity to maintain the integrity of theparticles, but must also have an appropriate ioniccomposition to minimize clumping . At the sametime, the conductivity of the buffer must conformto the requirements for regulation of current flowacross the chamber. These two limiting featuresrequire a rather extensive set of preliminary testsbefore optimal conditions can be defined for aparticular biological preparation .

A third limitation on the convenience of themethod is the need for some "internal marker"to which output fractions can be related . As incolumn chromatography, the whole output spec-trum may shift by three or four tubes because ofvariations in operating conditions from one runto another . This is illustrated by the differencebetween Fig . I and Fig . 13. This means that evenfor a well-studied spectrum it is necessary tomeasure either the protein content, turbidity, orother appropriate index of every output tube ineach experiment, in order to locate precisely theposition of the desired fraction .

Another advantage of the present method, apartfrom speed of preparation, is the opportunity whichit provides for accurate measurement of the sur-face electrical properties of separated particles .Other investigators (4, 5) have used electrophoreticmethods to prepare synaptic vesicles from thebrain but their techniques, although empiricallyuseful as preparative methods, have deficiencieswhich restrict the theoretical usefulness of theirmeasurements . For example, the mobilities ofsynaptosomes, synaptic vesicles, and mitochondriareported by Vos et al . (4) are in the same relativeorder as reported here, but their absolute valuesare only about one-half as large as ours . There areprobably three reasons for the discrepancy : (a)different buffer and temperature conditions wereused, (b) Vos et al . used a continuous sucrosedensity gradient which introduced unknownviscosity factors into the net mobility, and (c)electro-osmotic effects and electrode polariza-tion were ignored in their calculations . Thesefactors can be taken into account with the presentmethod, so that valid calculations of surface elec-trical properties of the various types of particlecan be made. It is interesting to note that thesurface charge density values in Table I are similar

in magnitude and sign to that calculated byAbramson et al. (30) for the guinea pig erythrocyte .Knowledge of the surface electrical propertiespermits one to develop theoretical approaches toan analysis of electrostatic interaction betweenparticles and membranes. An analysis of this typehas been undertaken (26), and will be publishedelsewhere .Further advantages of the present electro-

phoretic method over earlier ones include superiorregulation of temperature in the separation cham=ber, a shorter time requirement for separation,and continuous operation . These features raisethe possibility that purer preparations may beachieved by recycling individual fractions throughthe separation chamber . The potential advantagesare obvious. Attempts are currently being madeto apply this approach to the isolation of cellmembrane fragments .

The authors are indebted to Mr . T . Goodfellow, Mr .N. Rangaraj, Miss S .-W. Lee, Mrs. M. Mezari, andMiss M. Guttman for technical assistance . ProfessorP. Seeman, Mr. L. Pinteric, and Professor A. K .Sen provided valuable advice concerning electronmicroscopy and ATPase measurement .

Financial support for this work, including a fellow-ship to K. J. Ryan, was provided by the BickellFoundation, the Alcoholism and Drug AddictionResearch Foundation of Ontario, and the Medicaland National Research Councils of Canada.Received for publication 8 June 1970, and in revisedform 30 November 1970 .

REFERENCES

1 . WHITTAKER, V. P. 1965. The application ofsubcellular fractionation techniques to thestudy of brain function . Progr. Biophys. Mol.Biol. 15 :39 .

2. DE ROBERTIS, E. 1967 . Ultrastructure and cyto-chemistry of the synaptic region. Science(Washington) . 156:907.

3 . KADOTA, K., and T . KANASEKI . 1969 . Isolationof a synaptic vesicle fraction from guinea pigbrain with the use of DEAE-Sephadex columnchromatography and some of its properties .J. Biochem . (Tokyo) . 65:839.

4 . Vos, J., K. KURIYAMA, and E . ROBERTS . 1968 .Electrophoretic mobilities of brain subcellu-lar particles and binding of y-aminobutyricacid, acetylcholine, norepinephrine, and5-hydroxytryptamine. Brain Res . 9 :224.

5. SELLINGER, O. Z ., and R. N. BORENS . 1969 .Zonal density gradient electrophoresis of

RYAN, KALANT, AND THosAs Subcellular Particles from Guinea Pig Brain

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