Further evidence for a specific ganglioside fraction closely associated with myelin

BIOCHIMICA ET BIOPHYSICA ACTA

576

FURTHER

EVIDENCE

CLOSELY

ASSOCIATED

KUNIHII<(>

SUZUKI,

FOR A SPECIFIC WITH

JOSEPH

1;. PODUSLO*

Tile Saul R. fiovey Department of Newology, New Yavk, S. E’. (U.S..4 .) (Received

November

GANGLIOSIDE

FRACTION

MYELIN

AND SHIRLEP

Albert Eiwstei-~ Cd&v

E. PODUSLO of Medic&e,

r7th, 1967)

SUMMARY

I. Myelin was isolated from gray and white matter separately, and naturally the yield of myelin from white matter was much higher. The white matter myelin from rat brain contains most of the gangliosides found in myelin from whole rat brain, even though the gray matter myelin contains twice as much ganglioside per unit weight as white matter myelin. 2. Myelin isolated from either gray or white matter of adult bovine or rat brain had the characteristic myelin ganglioside pattern found previously for myelin prepared from adult rat whole brain; i.e. a predomjnan~e of the normal major monosialoganglioside G, (GniJ . 3. Myelin was prepared from the mixed homogenate of unlabeled adult rat brains and neonatal rat brains previously injected with n-[I-**C]glucosamine. Only 0.2-0.316 of the neonatal brain ganglioside was recovered in the myelin, and more than 99.6’?; of G, (GM*) in myelin derived from the adult brains. 4. The fatty acid compositions of the major nlonosialoganglioside G, (GMJ of whole gray matter, white matter and myelin were identical, with stearic acid comprising 90:; of the total fatty acids. 5. These findings show that the gangliosides in myelin from whole brain arise predominantly from white matter and are not due to random contamination by non-myelin tissue elements, particularly synaptic membranes. This study indicates the presence of a specific ganglioside fraction localized within the myelin sheath itself, or possibly in an axonal membrane closely associated with myelin.

Abbreviation : NANA, ~~-acet~lneuraminic acid. * Summer student, currently a third year student Wooster, Ohio.

in Chemistry

at the College of Wooster,

GANGLIOSIDES

ASSOCIATED WITH MYELIN

577

INTRODUCTION In our previous tained

from

constant

report

we demonstrated

brains

at various

rat whole

amount

of ganglioside

of KORET AND GONATAS’, of the myelin absolutely myelin

was

indeed

subcellular

experiments nature

after

designed

to tell

an intrinsic organelles.

to

of the ganglioside

provide

the purified stages

the normal

major

Since

whether myelin

in the myelin

approx.

the isolated

constituent

ob-

a relatively

myelin

carried

about

(G, percent

cannot

recovered

or derived

we have

information

fractions

90 molar

the ganglioside

In this study

further

myelin contained

monosialoganglioside

constituted

age 5 months3.

it was impossible

fraction

contaminating

and that

GM, of SVENNERHOLM~)

ganglioside

pure,

that

developmental

the

be

in our

from

other

out a series localization

of

and

fraction.

METHODS

Isolation of myeh, and analytical methods As in our previous experiments3, the improved PODUSLO AND SUZUKP was used to obtain tion

procedure

starting

have

been

sucrose gradient

was carried

did not exceed

that

contaminants

v/v)

that

The

dried

myelin

contained

small,

by centrifugation, and

vol.

the

chloroform

(2 : I, v/v).

The

upper

procedure upper

removed

phase,

procedure lower step

were

the trace to 3 davs. initial

once

The

tissue extraction

This

amount

in the upper The

and

dialyzed

tissue

initial

extract.

combined

upper

repeated

This

dissolved absolutely

present

in the

was an essential

of the contaminating the second at 4’

the dialysis

with

to eliminate was extended

the same way except (I

of gangpartition

for 2 days

was necessary

out with chloroform-methanol

of 0.2

and washing

to the amount

from

water

experiments, essentially

v/v),

once with

not

initially

amount

dialysis

tube.

combined

dried,

partition

easier.

phases

distilled

exhaustive

this

although

lipids

phase is large relative upper

(I:I,

washed

phases were

analysis

since the relative

In the isotopic

was carried

phase

partition, phase

is

the residue

of chloroform-methanol

and the above

of lower

was extracted

in myelin

to another To

: I,

proce-

phase by the addition

and the lower

against

residue

eliminating

was transferred

into the upper

combined

The rather

of sucrose.

whole

the

(2 : I, v/v),

experiments7,

samples.

insoluble After

(I

extraction

of chloroform-methanol

the final ganglioside

of water.

amount

with

was removed,

more.

present

concentrated changes

extract amount

double

to give the final concentration

phases. The

the small

in the isotope in myelin

frequent

upper

thus making

phase lipids

liosides

phase

tubes in order

of chloroform-methanol

impractical.

were partitioned

in chloroform-methanol repeated

essential,

a small combined

was added

FOLCH pure solvent once more

extraction

with

the

of tissue per centri-

the centrifuge

the chloroform-methanol

washes

Gangliosides

waters.

by the addition

the chloroform-methanol

was washed

centrifuged, extract,

because

When

on the discontinuous

so that the amount

We did not use our standard

the double

of the isola-

in full shortly.

centrifugation

not to overload

of NORTON,

at a minimum.

was extracted

59/o water.

making

residue

be kept

procedure

The essentials

be published

out in two batches

5 g. It is critical

would

dure for ganglioside6,

The

and will

isolation

fractions.

brain tissue was more than 5 g, the initial

fugation

very

outlined3

myelin

that

the

: I, v/v) containing

no water. Biochim.

Biophvs.

Acta,

152 (1968) 576-586

Ii. SUZUKI, J. F. PODUSLO, S. E. PODUSLO

578

The total N-acetylneuraminic acid (NANA) was estimated by the resorcinol method of SVENNERHOLM* as modified by MIETTINEN AND TAKKI-LUUKKAINEN~. Ganglioside patterns were determined as described previously5110. The ascending solvent system of chloroform-methanol-z.5 M ammonia (60 :40:9, v/v/v) was used for the thin-layer chromatography instead of the n-propanol-water system. The chromatography was run in a paper-lined tank, then desiccated for at least 3 h, and re-run once more in the same tank. The radioactivity of individual gangliosides was determined as we described recently?. The lower phase lipids were analyzed for total cholesterolll, total phospholipids12, and total galactolipids13. Individual phospholipids were analyzed

after

the thin-layer

chromatographic

separation

of the total

lipid

mixture14115. Cerebroside and sulfatide were determined by the same orcinol method for total galactolipidsl3 after silicic acid column chromatography of the total lipid mixturelG. The fatty ganglioside

acid composition

G, (Ghll) obtained

was determined

on the normal

from adult rat gray matter,

major

white matter,

monosialoand from

two separate rat myelin preparations. The ganglioside was isolated in pure form by the preparative thin-layer chromatography on silica gel HR plates in the solvent system described above. Methanolysis was carried out directly in borontrifluoridemethanol reagent (Applied Science, State College, Pa.) at 100’ for 90 min under nitrogenl’. The fatty acid methyl esters were isolated and purified as described by MORRISON AND SMITH’~, and analyzed

by gas-liquid

chromatography.

Experimental design Gray and white matter myelin. Duplicate experiments were carried out on both fresh bovine and adult rat brains. In the bovine experiments, myelin fractions were prepared from cortical gray matter respectively. In rat-brain experiments,

and from centrum semiovale white matter, 20 adult rats were used for one experiment.

Cortical gray matter was pooled as the gray matter sample, and the portion of brain stem below pons and above the upper cervical cord was collected as the white matter sample. Therefore, both gray and white matter samples of the rat brain were less cleanly separated from each other than those of bovine-brain experiments. Samples of whole gray and white matter were included in each experiment and analyzed for gangliosides simultaneously with the myelin fractions. The myelin fractions from gray matter were also analyzed for total cholesterol, total and individualphospholipids, and total and individual galactolipids. Isotope dilution experimed. This experiment was designed to provide a starting brain homogenate for myelin preparation, which contains radioactivily labeled ganglioside and unlabeled myelin. The amount of labeled ganglioside in the myelin fraction would then be the direct indication of the impurity of myelin. Brain ganglioside can be labeled to a relatively high specific activity by injecting D-[I-14C]glucosamine into neonatal rats. The rat brain at this developmental stage does not contain myelin. There is no histologically demonstrable myelin in neonatal rats. During the developmental study of rat myelin composition, we observed that it was impossible to prepare any myelin from rat brains younger than IO days. In the same study3, the extrapolation of the curve of the myelin yield zleysus age indicated zero myelin around age 12 days. Therefore, the homogenate of isotope-injected neonatal rat brains and Biochim.

Biophvs.

Aicta, 152 (1968)

576-586

GANGLIOSIDES ASSOCIATED WITH MYELIN

579

unlabeled adult rat brains should contain labeled ganglioside and myelin which is completely free from radioactivity. ~-~I-l~~~Glucos~ine (specific activity, 10.8 mC~mmole), purchased from New England Nuclear Corporation, Mass. was dissoIved in physiological saline, 20 ,uC/o.r ml, and injected subcutaneously into rg neonatal rats, age 2 days, at the level of I ,uC/g body weight. The injected neonatal rats were killed after 24 h. 4 adult rats were also killed, and the animals were divided into 3 groups: (I) 7 neonatal rats, (2) I adult and 3 neonatal rats, and (3) 3 adult and g neonatal rats. The brains of individual groups were pooled. Groups I and z were extracted and analyzed for gangliosides, and ganglioside radioactivity measured. The brains of the 3rd group were homogenized, and the myelin fraction prepared. The isolated myelin was analyzed for the content and the radioactivity of gangliosides. Since the radioactive contamination due to nucleotide sugars was insignificant under the labeling condition described, the snake venom pI~osphodiesterase treatment of the samples was unnecessary’. RESULTS

Gray and white matter myelin. Yield and ga?zglioside content (Table I). On unit fresh weight basis, the yield of myelin from gray matter was 6 and SO,i,of that from white matter in bovine brain experiments, and 14 and 2r”,6 in rat brain experiments. A higher yield of myelin from gray matter in rat brain experiments was expected, because the gray matter samples contained a small amount of white matter as contaminant. The generally low yield of myelin in bovine Exp. I is probably due to the fact that we used the Spinco SW-25.1 rotor for centrifugation instead of the larger capacity SW-252 that was used for the other experiments. The total ganglioside NANA recovered in the TABLE

I

GANGLIOSIDES ____-

IN WHOLE

Species

Beef, Expt.

1

White: Gray

Beef, Expt.

I

Expt.

I

Expt.

z

: :

White: Gray:

Rat,

:

White Gray

Rat.

TISSUE

AND

White Gray:

ISOLATED

MYELIN

Starting wet ZeJt. isi

k’ieZd of my&n* (ms;i

whole

2.43

-

myelin whole myelin whole myelin whole myelin whole my&in whole

5.20 1.11 5.19 I.49 8.36

43.25 2.56 92.66

Sample

I.80

9.27

7.42

1.78

-

5.89 1.33

51.89 -

myelin

8.08 1.23

7.29

myelin whole

::::

: whole

67.93 -

219

-

8;:

39 -

3 213 42 99=

110

$31;

-

4.5 88

31 1013

60 -

8.4 548

115 -

47 105~

69 -

my&n 7-35 14.69 13.7 94 __ ___._.-._ * Yield of myelin per g wet wt. ** NANA per g wet wt. The values for my&n fractions represent the amounts of NANA myelin fractions obtained from 1 gram of wet tissue. *** NANA per IOO mg dry myelin. Biochim. BiopFzvs. Acfa,

152

(1968)

in the

576-586

K. SUZUKI,

fractions

my&n

from gray matter,

of that from white matter experiments. The percentages fraction

J. F. PODUSLO,

S. E. PODUSLO

again on unit fresh weight basis, was

for the bovine

experiments,

of the total tissue ganglioside

were 7.8 and 17.30/b for bovine

18

and 17:<

and 27 and 2970 for the rat NANA recovered

white matter,

5.3 and 8.6:;;

in the myelin for rat white

matter, 0.4 and 0.7”/0 for bovine gray matter, and 0.8 and 1.376 for rat gray matter. Despite the high ganglioside content of gray matter, Norton’s myelin preparation procedure effectively eliminates almost all ganglioside from gray matter myelin. On the other hand, although the total ganglioside content of white matter is low, a portion

significant

of white matter

ganglioside

is recovered

in the myelin

fraction.

The ganglioside content of the myelin preparations from white matter was within the range of the previously reported values for bovine myelin15y1s and for rat myelins. Myelin from gray matter,

however, contains

as white matter myelin. Ganglioside patterns (Table II, Fig. of the normal major monosialoganglioside firmed matter

approximately

twice as much ganglioside

The previously reported3 predominance G, (G& in the myelin fraction was con-

I).

in both adult rat and bovine myelin. Not only did the myelin from white show this characteristic ganglioside pattern, but the myelin from gray matter

also had essentially

the same distribution

of the individual

gangliosides.

The gang-

lioside patterns are expressed here as percentage distribution of NANA rather than molar distribution, because some of the slower moving multisialogangliosides were determined together, thus making the calculation of the data on a molar basis theoreticallyimpossible. In bovine myelin, approximately half of the total ganglioside NANA is in G, (GDI1), and in rat brain myelin, 70 to 809,; of the total NANA is in G, (GnfJ. It should be noted that, TABLE

despite the probable

GANGLIOSIDES

Nomenclature of eanelioside is that HOLM nomenclature in parenthese@. ”

of KOREY

AND GONATAS~ with

Percent

Sal?lplP

Species

GI (CT,)

Beef,

Rat,

Expt.

Expt.

Expt.

I

2

I

White

Eupt.

2

:

Gray White

:

Gray

:

:

White Gray

Rat,

: :

White

Gray

: :

Biophvs.

Acfa,

of

distributiow G,

(G&b)

the corresponding

(GDJ

G, fG.vd

36.7 31.1

35.4

whole whole

35.2 42.1

53.7 33.6

27.9 48.0 II.1 24.0

myelin whole

5i.7 36.4

50.2

48.2 13.2

19.9

52.7 19.3

36.1

69.1 18.1

?.0.g

myelin whole

45.8 60.4 L___--_

myelin whole

23.9 45.0

myclin whole

‘5.5 25.1

24.1

83.0 19.8

24.4 ‘5.9

45.0

70.7 10.2

30.g v-

myelin whole

26.9

152 (1965)

16.3 ____57(i-556

-_

SVEXNER-

NAN,4 G,

whole myelin

myelin Biochinz.

myelin as

II

DISTRIBUTIONOF THE INDIVIDUAL

Beef,

lower purity of gray matter

___~

So.0

GANGLIOSIDES

ASSOCIATED

WITH

WHOLE

MYELIN

WHOLE

MYEL I N

MYELIN ..

WHITE

GRAY

Fig. I, Thin-layer chromatogram of the ganglioside patterns of rat whole gray and white matter and their respective myelin preparations. Solvent System: chloroform-methanol-z.5 M ammonia (60 : 40: 9, v/v/v) ascending. Spots were located by the resorcinol spray for sialic acid. Ganglioside nomenclature is that of KOREYAND GONATAS’with the corresponding SVENNERHOLM nomencla-

ture in parenthw&. indicated

by its high ganglioside

content,

the same, if not higher prepondrance

Nature

of gray-matter from gray4 matter,

fractions

the normal major monosialoganglioside

in these gray matter

has

fractions.

myelin. Under the electron microscope, the myelin both bovine and rat, appeared to consist largely of

reasonably well-preserved myelin lamellae. found as contaminants. No other identifiable

Lipofucsin-like bodies were occasionally subcellular components, such as synaptic

elements or mitochondria, were found. These fractions were obviously less pure than the myelin prepared from white matter, where contaminating subcellular components were extremely difficult to find. Another indication that the gray matter myelin is in fact mostly myelin is given by the analytical data of these fractions (Table III). The distribution of the major components and lipid composition of both bovine and rat gray matter myelin are almost identical with those of myelin from white matter or TABLE

III

COMPOSITION

OF

VARIOUS

MYELIN

Constifrtrnts

FRACTIONS

Bovine

brain m_yelin

White

Gva?, I

Gray II

-

8.2 23.8 67.2

Rat brain

6.3. 30.5 59.7

Phospholipids ethanolamine lecithin

phospholipids

sphingomyelin monophosphoinositide serine phospholipids Glycolipids cerebroside sulfatide * Data

presented

by NORTON,

0.4 26.9 70.8

0.7 27.2

68.5

T/b lipids

9: lipids Cholesterol

Gra_v

“/b dry zelt.

3; drv wt. Insoluble residue Proteolipid protein Lipids

myelin

Whole brain*

27.9 41.5 ‘4.4

28.0

31.8

42.3 13.0

12.5

12.9

42.4 12.8 12.7

8.7 0.7

8.5 I.1

3.7 28.0 23.3

4.6 23.3

4.0

3.6

9.0 0.9 5.0 24.0

20.2

27.4 39.3 ‘4.9 9.6 3.0 0.9 6.7 28.4 21.2

7.3

26.7 37.9 15.3 13.7 3.0 0.9

2.2 25.4 19.4 4.1

PODUSLOAND SUZUKI~. B&him.

Biophys.

Acta,

152 (1968)

576-586

K. SUZUKI, J. F. PODUSLO, S. E. PODUSLO

582 whole brain. Total gray matter

contains

more than 300/Oinsoluble

residue, only a few

percent of proteolipid protein, and less than 35% lipids. Close to 700/b of the total lipid in whole gray matter is phospholipids and less than 10% glycolipids. Although the higher ganglioside content might be indicative of higher contamination, these morphological and analytical data, nevertheless, present strong evidence fraction prepared from gray matter is still mostly myelin.

that themyelin

Isotope dilution experiment In this experiment, the amount of radioactive ganglioside in the myelin fraction indicates the amount of non-myelin contaminants derived from neonatal brains. The data

presented

TABLE ISOTOPE

in Table

IV permit

various

calculations

and lead to the following

IV DILUTION

EXPERIMENT

Neonatal rats received subcutaneous injection of n-[I-i4C]glucosamine are unlabeled. Nomenclature of ganglioside as Table II.

Number of animals Fresh wt. (g) neonatal adult Weight of my&n (mg) Total NANA @g) Ganglioside G, (GT~). Spec. Pattern* activity** G, (Garb), Pattern* _ Spec. activity** G, (GD,,), Pattern* Spec. activity** G, (GM~), Pattern* Spec. activity**

h earlier. Adult rats

24

_ ..~_

IB8ole brains

Whole brains

Myelin

7 neonatal

3 neonatal + I adult I.23

9 neonatal + 3 adult 3.64 5.93 160.89 137

2.90

2.04

2083

roo9

1.~9 39.2 ‘9.7 r4o 34.9 165 6.2 206

30.3 3r 1 26.4

-_____

22

38.8 (Pattern*) 5.6 (Spec. activity**)

31.5 4’ i II.9 25

58.3 0.7

* Percent distribution of NANA. ** Counts/min//lg LLANA.

conclusions.

The total radioactivity

of gangliosides

in the g neonatal

rat brains added

to the 3 adult brains for the preparation of myelin can be estimated either from the 7 neonatal brain sample or from the 3 neonatal and I adult brain sample by using the total NANA, the percentage distribution of NANA among individual gangliosides, and the specific activity of each ganglioside. The total radioactivity in 9 neonatal brains thus calculated is 1.98.10~counts/min from the data of the sample of 7 neonatal brains, and 1.94’105 counts/min from the mixed homogenate sample. Therefore, we consider that gangliosides containing a total of 1.96-105counts/min were added to the 3 adult brains for myelin preparation. Similarly, the total radioactivity of the normal major monosialoganglioside G, (GMJ added for myelin preparation was calculated to be 1.67. IO” counts/min and 1.86.10~ counts/min respectively, with the average of 1.77.10~counts/min. From the total NANA, the specific activity, and the pattern of gangliosides in myelin, we can calculate the total radioactivity of ganglioside recovered in myelin to be 353 counts/min, and that of G, (Gsrl) to be 56 counts/ min. Therefore,

we can conclude

that

Biochim. Bioph?,s. Acta, 152 (1968) 576-586

less than o.z”//oof total

ganglioside

initially

GANGLIOSIDES

present

ASSOCIATED

in the 9 neonatal

recovery

583

WITH MYELIN

brains was recovered

in the myelin fraction.

of the labeled G, (GMJ in the myelin fraction

added. These figures attest

to the general effectiveness

dure in eliminating non-myelin components The differences in specific activities

is

0.3%

Similarly,

of the myelin isolation

of the brain. of individual

the

of the amount initially

gangliosides

proce-

indicate

the

degree of the dilution of labeled gangliosides

at each step. In the mixed homogenate, ZZ”/~of G, (GT~), 16% of G, (Gmb), 25% of G, (GD~,), and 12’3, of G, (GMJ were derived from the neonatal brains. The different degrees of dilution for each ganglioside were due to the different ganglioside patterns in neonatal and adult rat brainslg. Gangliosides,

G,-G,

(GT,-Gnla)

in the myelin

fraction

prepared

from such mixed

homo-

genate were further diluted approx. 6-fold, and the dilution for G, (GMJ from the starting homogenate to myelin was 35-fold. Comparing the specific activities of the starting neonatal brain gangliosides and those in myelin, we can conclude that 3 to 40/x of G,-G,

(Gri-Gnl,)

in myelin originated

in the added neonatal

brains,

and only

0.3 to 0.4% of G, (GNIJ in myelin derived from the neonatal brains. Thus, this experiment unequivocally showed that almost all ganglioside in the myelin fraction, particularly

G, (GHJ, originated

in the adult brain.

Fatty acid composition The fatty acid compositions

of the normal major monosialoganglioside

G, (GM,)

obtained from gray matter, white matter, and isolated myelin are virtually identical, with stearic acid (18:o) comprising 90% of the total fatty acids in all samples (Table V). Predominance of stearic acid in brain gangliosides is a well-established fact (refs. 20-23).

The normal

major

monosialoganglioside

in the myelin

fraction

is not

specific in this regard.

TABLE FATTY

\ ACIDS

OI?

NORMAL

MAJOR

MONOSIALOGANGLIOSIDE

Values are expressed in weight percentage of the total fatty acids. The normal major monosialo-

ganglioside G, (GMI) obtained from gray matter, white matter and from two separate preparations of isolated mpelin was analyzed for the fatty acid composition. Because of the limited amounts of the sample available for analysis, the data except for 18 : o and 20: o must be considered to have margin of error * 509:. -. My&n IT Fatty acids M_velin I Gra_v matter White matter 14:o 15:o 16:o 17:o 18:1 18:o 1g:o 20:1 2o:o 21: I 21 :o 22: I 22:o 23:o 24:1 24:o

2.2 91.6 o..? 4.9 0.3 0.5 Trace 2 Trace

Trace Trace 2.4 Trace 91.8 0.4 Trace 4.5 0.2 Trace 0.7 0.8 0.3

1.6 0.5 Trace 92.0 0.4 Trace 3.2 Trace 0.6 -

Trace Trace 2.6 Trace 89.0 0.3 Trace 3.9 Trace Trace

0.3 Trace Trace Trace

0.4 1.2 I.5

0.1 0.2

I.2

Biochim. Biophys. rlcta,

152

(1968) 576-586

K. SUZUKI,

$34

J, F. PODUSLO,

S. E. PODL’SLO

DISCUSSION

In our previous repor+, we did not know the proportion and the pattern of ganglioside in the myelin fraction derived either from gray or white matter, since we studied myelin prepared from whole brains. We have now obtained the answer to the above question by the first series of experiments on separate gray and white matter. If we make an assumption that the whole brain contains approximately equal amounts of gray and white matter, the bulk of whole brain myelin naturally derives from white matter. So::; or more of total ganglioside found in whole brain myelin originates in white matter, despite the much higher ganglioside covery of whole tissue ganglioside in myelin fractions

content of gray matter. The rewas consequently IO times higher

from white than from gray matter. The myelin fraction obtained from gray matter appears to be mostly myelin, judged by the electron microscopic and analytical data. Furthermore, the characteristic preponderance of G, (Gary}-ganglioside was found in both myelin fractions from gray and white matter. This finding also escludes the synaptic membrane as the possible contaminant in our myelin fraction as we initially suspected3, because synapses are far more abundant in gray matter. The electron microscopic examination also failed to detect synaptic elements. Our myelin isolation procedure appears to eliminate synaptic membrane quite effectively despite its similar density

to myelin2”. If most of the ganglioside in myelin fraction derives from white matter, it must be localized either in myelin, which is an extension of the oligodendroglial cell mem-

brane,

or in neuronal

constituent

most abundant

in white matter,

the axonal

mem-

brane. We tried to narrow down this question further by the isotope dilution esperiment. The rationale behind this experiment is that only the ganglioside of the neonatal brains are labeled, the neonatal brain has no my&n but does have all other brain constituents, and that adult brains contain no radioactivity. Therefore, if the myelin prepared from the mixed homogenate of neonatal and adult brains is 100:~ pure, it should not contain any radioactivity. On the other hand, any radioactivity found in the myelin fraction must be considered due to contamination by nonmyelin components derived from neonatal brains. The results of this experiment clearly showed that the labeled gangliosides of the added neonatal rat brains were almost completely eliminated from the myelin fraction. Better than oo.Ono of the predominant G, (Gn,)-ganglioside in myelin originated in the adult brains. If we assume that the properties of the axonal membrane do not change during development, then the results of this experiment exclude free-floating axonal fragments as the possible contaminant. Three possibihties now appear to remain as the site of localization of gangliosides; (I) myelin sheath itself, (2) adult axonal membrane which has the properties different from those of neonatal rat, and (3) axonal membrane which is tightly bound to the myelin lamellae ill s&f, and may be isolated together with myelin. The last possibility seems to us improbable because of the relatively constant amount of ganglioside per unit weight of myelin throughout the developmental stages3. The bulk of myehn increases drastically during active lnyelinati~~n, but the amount of the bound axonal membrane is not likely to increase at the same rate. In fact it should remain relatively constant. The second possibility cannot be ruled out by this series of experiments. The adult axonal membrane conceivably has different properties so that it contaminates the myelin fraction whereas the neonatal

GANGLIOSIDES ASSOCIATED WITH MYELIN axonal

membrane

functionally

is excluded.

closely associated

585

Such changes

could be either merely

with the process of myelination.

coincidental

The latter

or

possibility

seems more probable because the amount of gangliosides in the myelin fraction increases at the same rate as myelin during development. But no clear-cut morphological difference is observed in the axonal membrane before and after myelination, and we are inclined to think that the possibility membrane

of the changing property

is less likely than that of the presence

of ganglioside

of the axonal

in the myelin sheath

itself. If a myelin fraction can be prepared which satisfies all the ultrastructural and chemical criteria of myelin but is completely free of ganglioside, the above question would be readily solved. However, completely ganglioside-free myelin has not been obtained. THOMPSON, GOODWIX AND CUMINGS~~prepared myelin from human brains by both sucrose and cesium chloride density gradient and stated that myelin contains no ganglioside. We have been unable to confirm their finding, and we consider that the difference may be due to the relatively insensitive analytical method they used for gangliosides. They analyzed the total chloroform-methanol extract for ganglioside. The level of ganglioside we find in myelin is so low compared to other lipids (50 l&g NANA ~1s. 70 mg lipid per IOO mg myelin) that reliable analysis is difficult without properly separating ganglioside from other lipids. Therefore, although their isolation procedure may indeed yield ganglioside-free myelin, we remain unconvinced until their finding is confirmed by more sensitive analytical methods. We found 30 to 5opg of ganglioside NANA per IOO mg myelin prepared from two normal human brains, ages 2.5 and 5.5 years, and from two cases of spongy degeneration of white matter, all post-mortem

frozen specimens.

In these cases, the normal major

lioside G, (GnIJ was again predominant

(5

80”;)

monosialogang-

in myelinZe.

ACKNOWLEDGEMEKTS Dr. KINUKO SUZUKI, Department

of Pathology

(Neuropathology),

ried out the electron microscopic examination of various myelin investigation was supported by grants, R-160-67C from the United Foundation,

and NB-03356

from the U.S. Public

Health

kindly car-

fractions. Cerebral

This Palsy

Service.

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