Acetyl-CoA carboxylase in rat brain. I. Activities in homogenates and isolated fractions

DevelopmentalBrain Research, 43 (1988) 123-130 Elsevier

123

BRD 50802

Acetyl-CoA carboxylase in rat brain. I. Activities in homogenates and isolated fractions Francine A. Tansey and Wendy Cammer Departments of Neurology and Neuroscience, Albert Einstein Collegeof Medicine, Bronx, NY 10461 (U.S.A.) (Accepted 11 May 1988)

Key words:Acetyl-coenzyme A carboxylase; Oligodendroglia; Myelination; Rat brain

Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting and/or first committed step in fatty acid biosynthesis. Because fatty acids must be synthesized as components of the galactolipids and phospholipids in myelin, high specific activities of ACC would be expected in brain during myelination and in the myelinating cells, the oligondendroglia, in particular. Under reaction conditions where ACC was linear with time and protein concentration, we found specific activities of 1.7 and 3.1 nmol/min/mg protein in supernatants from forebrains and brainstems, respectively, of 20-day-old rats. In both regions, ACC declined during development, particularly after the age of 20 days. To separate forebrain into discrete fractions containing cells, membrane vesicles, and other components, without destroying the ACC, it was necessary to modify the published methods by adding citrate to the isolation medium and by omitting trypsin. A fraction which sedimented over 1.2 M sucrose showed the highest specific activities and recoveries of ACC. This fraction was rich in small cells, many of which immunostained with antibodies against galactocerebroside and carbonic anhydrase, both of which are localized in oligodendrocytes and immature glial cells. The cells in this fraction also immunostained with antibodies against ACC. The resuits are consistent with the hypothesis that ACC is an oligodendrocyte-associated enzyme, although it probably is not exclusive to cells of that type.

INTRODUCTION There is now strong evidence that fatty acids can be synthesized de novo in brain, via a c e t y l - C o A derived either from acetate or from k e t o n e bodies 2'4's' 10.11,15 The first c o m m i t t e d step in the sequence yielding fatty acids from a c e t y l - C o A is catalyzed by a c e t y l - C o A carboxylase ( E C 6.4.1.2) ( A C C ) , a cytoplasmic enzyme which converts a c e t y l - C o A plus bicarbonate to m a l o n y l - C o A (reviewed recently in ref. 14). The results of some of the few studies, where A C C was m e a s u r e d in CNS tissue in vitro, suggest that A C C could be rate-limiting for fatty acid synthesis in brain, particularly at limiting concentrations of bicarbonate 3,5,11. Because of the r e q u i r e m e n t for fatty acids as components of the galactosphingolipids and phospholipids in myelin, one would expect to find particularly

high specific activities of A C C in brain during myelination, and also to find significant specific activities in the myelinating cells, the oligodendroglia. While values in the literature do suggest a developmental decline in A C C activity as the rate of myelination diminishes, there are discrepancies among the actual values 5A1,16. This study was begun with the goals of: (1) setting up the assay for A C C in CNS tissue to obtain reproducible data; (2) in view of the discrepancies, to reevaluate the specific activities in developing brains; and (3) to see w h e t h e r A C C might indeed be associated with the oligodendrocytes. MATERIALS AND METHODS S p r a g u e - D a w l e y rats were purchased from C a m m (Wayne, N J,) and Charles River, (Wilmington, M A ) . U n l a b e l e d substrates and cofactors were pur-

Correspondence: W. Cammer, Albert Einstein College of Medicine, Department of Neurology, F-140, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A. 0165-3806/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

124 chased from Sigma Chemical Co. (St. Louis, MO): '4C-labeled bicarbonate, from New England Nuclear (Boston, MA): reagents, other than primary antisera, for immunocytochemistry, from Accurate (Westbury, NY); and sucrose and salts from Sigma and other conventional vendors. The antisera, all raised in rabbit, were obtained as follows: anti-galactocerebroside (GC) from Dr. C. Raine (Bronx, NY), antihuman carbonic anhydrase C (CA) from Calbiochem Behring (San Diego, CA), and anti-rat ACC from Dr. K.G. Thampy (Houston, TX). ACC was assayed by the conventional method, which involves measuring the incorporation of 14C from H14C03 into non-volatile counts ~'. Samples were preincubated 30 rain at 37 °C in the presence of 47 mM Tris-HCl, pH 7.2, 21 mM potassium citrate, 21 mM MgC12, 0.5 mg/ml bovine serum albumin (BSA), and 1.1 mM dithiothreitol (DTT). After preincubation, the samples were added to the substrates, to give final concentrations in the reaction mixtures as follows: 118 mM Tris-HC1, pH 7.2, 24 mM potassium citrate, 24 mM MgCI> 0.23 mM acetyl-CoA, 5.8 mM ATP, 0.6 mg/ml BSA, 1.2 mM DTT, and 23 mM NaHlaCO3 (50 mCi/mmol). We found this concentration of bicarbonate to be saturating, in the presence of these concentrations of the cofactors and the other substrates, and used it, accordingly, to optimize the counts per mmol in the substrate while conserving isotope. The reaction tubes were incubated 0, 2, 4, and 6 rain at 37 °C, after which the reaction was stopped by adding 0.25 vols, each of concentrated HCI and methanol. The acidified reaction mixtures were evaporated to dryness, and 14C-contents were determined by scintillation counting. Specific activities, in nmol/min/mg protein, were calculated using standard curves (counts per min versus nmol HCO3) obtained for each experiment, using aliquots of the labeled substrate. As expected, the reaction was inhibited by over 80% in the presence of avidin at 100,ug/ml or malonyl-CoA at 0.13 mM and required the 30-rnin preincubation in the presence of citrate and other reagents, as described above. When high-speed supernatant fractions were prepared, as described below, and assayed at appropriate protein concentrations, the ACC reaction was linear with time and protein concentration for at least 6 min (Fig. 1). Incorporation was always measured in duplicate at t,, and the 3

6

O c~

57 /

4-

O u -r

B 2

"6 E

1

0

1

2 3 4 Time in minutes

5

6

Fig. 1. Time course of the acetyl-CoAcarboxylase reaction catalyzed by high speed supernatant fractions from rat forebrain and brainstems. The enzyme assay was performed as described in the Materialsand Methods section. 20 d BS = brainstem from 20-day-oldrats, 275/~gprotein per tube, open circles; 20 d FB = forebrain from 20-day-oldrats, 272 ,ug proteinper tube, triangles; 60 d BS = brainstem from 60-day-old rats, squares, 382 ¢tg protein per tube, and closed circles, 230 ~g protein per tube. All the t o points were at 0 to 0.33 nmol. At times where only one point is shown, the values for the duplicate tubes were identical.

reaction times, using -- 250ktg protein per tube. High-speed supernatants were prepared by homogenizing whole tissue, minced tissue, screened tissue or cells in 10 mM Tris-HCl," pH 7.2, centrifuging 30 rain at 25,000 g, and then centrifuging the resulting supernatants 60 rain at 100,000 g. The supernatants were frozen and lyophilized and stored at -20 °C, where they retained their activity for several weeks. The data shown below all represent assays performed within one week of preparation. There was no change in 14C-fixation by the brain supernatants after passage through Sephadex; therefore, as in studies of ACC in brain in several other laboratories TM, Sephadex chromatography was not performed before obtaining the data reported below. RESULTS The ACC specific activities in high-speed supernatants from two regions of the rat brain are shown in Table I. While the specific activities in the soluble fractions were reproducible, those measured in

125 TABLE I Acetyl-CoA carboxylase in rat brain supernatants Supernatants were prepared and ACC assays performed as described in the Materials and Methods section, Values from the literature are also shown in order to permit comparisons. To convert units per gram wet weight to units per mg protein we used values for mg supernatant protein per gram wet weight determined in our laboratory. Region

Specific activities (nmol/min/mg prot. ) Age (days)

This study

Patel and Tonokow 11.

Gross and Warshaws*

Yeh et al. 16

Forebrain

10 15 20 60

2.32 + 0.35 (3) 1.80 + 0.27 (4) 1.68 + 0.27 (3) 0.95, 0.81

0.59 0.55 0.57 0.51

2.9 2.4 2.0 1.7

2.5 1.8 1.9 1.9

Brainstem

10 20 60

3.16 + 0.87 (5) 3.11 + 1.57 (6) 1.81 + 0.22 (4)

-

-

0.74 0.49 0.63

* Values are soluble fractions from whole brains.

w h o l e h o m o g e n a t e s w e r e v a r i a b l e , the t i m e - c o u r s e s not consistently linear, and the v a l u e s o n l y o n e - s i x t h

o l i g o d e n d r o c y t e s f r o m the r e m a i n i n g tissue c o m p o nents 12. H o w e v e r , b e c a u s e A C C is v e r y sensitive to

TM, it

to o n e - t h i r d t h o s e in s u p e r n a t a n t s . T h e assay m e t h o d

p r o t e o l y t i c e n z y m e s and to o x i d a t i o n

is in fact m o s t suited for A C C - e n r i c h e d s o l u b l e super-

possible to retain e n z y m e activity w h e n that m e t h o d

natant fractions r a t h e r t h a n w h o l e tissue 6. T h e r e -

was

fore, o n l y the v a l u e s for h i g h - s p e e d s u p e r n a t a n t frac-

m e t h o d , i n a c t i v a t e d A C C , and e v e n w h e n the trypsi-

used.

Trypsinization,

as

described

was n o t in

that

tions o b t a i n e d f r o m dissociated tissue and f r o m the

nization step was o m i t t e d ,

isolated cells and vesicles will be s h o w n below.

t h r o u g h n y l o n a n d / o r steel screens r e s u l t e d in signifi-

passage of the tissue

T h e m o s t practical o f the a v a i l a b l e m e t h o d s for iso-

cant loss o f e n z y m e activity. A d d i t i o n o f D T T , fatty

lating cells f r o m rat f o r e b r a i n s uses a n e u t r a l bal-

a c i d - f l e e B S A , and p r o t e i n a s e inhibitors to the isola-

anced salt s o l u t i o n , in which t h e n e u r o n s and astro-

tion m e d i u m did n o t solve this p r o b l e m . W h e n we ho-

cytes are lysed, f o l l o w e d by t r y p s i n i z a t i o n and pas-

m o g e n i z e d the tissue, r a t h e r t h a n s c r e e n i n g it, the

sage t h r o u g h steel and n y l o n screens to dissociate the

A C C was p r e s e r v e d ; h o w e v e r , a f t e r h o m o g e n i z a t i o n

14 ml

~_~

10ml

~j]

9ml

A or A'

20rain 3300 xg

sw28

B of Bi

D or D'

EorE'

C

ore

I

Fig. 2. Sedimentation of fractions from forebrains of 15-day-old rats. Fifteen- to 16-day-old rats were decapitated and the forebrains were dissected out, weighed, and minced, at 4 °C, in a medium containing 75 mM Tris-HC1, pH 7.2, 30 mM potassium citrate, 31 mM MgCI:, 0.8 mg/ml bovine serum albumin, and 0.23 mg/ml dithiothreitol. The osmolality was measured at 299 mOs, compared to 310 mOs in the medium used by Snyder et a1.12. Under these conditions the neurons and astrocytes would lyse 12. The minced forebrains were incubated for 1 h ~t 4 °C to permit equilibration with the citrate in the medium, and were then passed through a nylon mesh (145 /am) one time with gentle pressure and then through a stainless steel mesh (74/am) 3 times with gentle suction. The sucrose concentration was adjusted to 0.85 M or 1.0 M by slow addition of 1.7 M or 2.0 M sucrose, and the suspension (-- 80 ml total) was layered over the sucrose density gr~idient shown on the left side of the figure. The tubes were centrifuged and their contents processed further as shown in the figure (Norton and Poduslo 9, somewhat modified). If the least dense sucrose solution was 1.0 M, the fractions were designated A - E , and, if 0.85 M, A ' - E ' . The fractions were pelleted by centrifuging 10 min at 700 g and were frozen overnight. After thawing, high-speed supernatants were prepared as described in the Materials and Methods section. The brains from 10 rats were processed in six 33 ml centrifuge tubes during the first density gradient centrifugation step.

12f~ we could not separate cells, blood vessels, m e m b r a n e

myelin contents may have interfered with disaggre-

vesicles, etc., on the density gradient. Thus, it became necessary to change the cell isola-

gation of the tissue in the absence of trypsin. It should

tion method in order to preserve enzyme activity. Al-

be noted that both centrifugation steps were necessary for optimal separation of the 3 fractions. Possi-

though the changes, to be described below, resulted in differences from the findings of Snyder et al. ]2.

loading the first gradient and pipetting onto the sec-

with respect to the distribution of fractions on the density gradient, it was possible to characterize the

ond gradient. W h e n the uppermost layer of the first gradient

fractions in whch A C C was recovered.

bly, some clumps of cells were dispersed while un-

contained 1.0 M sucrose, the soluble supernatant

The inactivation by trypsin and/or screening had

prepared from the medium-density fraction, which

suggested that plasma m e m b r a n e s might break and reseal during disaggregation of the tissue. Therefore

sedimented through the top layer and floated on 1.2

we added to the isolation m e d i u m reagents known to promote and protect the active, relatively aggregated form of A C C (e.g. see ref. 6) (legend to Fig. 2). If this m e d i u m was used during screening, much of the A C C survived. The optimal conditions for isolation are shown in Fig. 2 and the A C C activities, in Table II. The best separations were obtained using forebrains from 15- to 16-day-old rats. Tissue from older animals or from brainstems did not distribute so well on the gradient, probably because the higher

riched over that prepared from the screened tissue.

M sucrose (fraction B in Table II), was slightly enwith respect to A C C specific activity, although not to an extent that would be statistically significant. On the average, 68% of the A C C recovered from the gradient was in that fraction. W h e n the uppermost layer contained 0.85 M sucrose, more of the A C C (92%) was recovered in the medium-density fraction (B'), and the specific activity in the supernatant from that fraction was somewhat lower, at 1.t4 nmol/

T

TABLE II

1.4

A CC specific activities in fractions from the density gradients

Fractions were prepared from forebrains of 15-day-old rats as shown in Fig. 2, and supernatants were prepared from those fractions and ACC specific activities determined, as described in the Materials and Methods section. In 3 experiments, 1.0 M sucrose was used as the top layer in the first gradient, and in 3 other experiments 0.85 M sucrose was used. Mean values are shown, with standard errors and with the number of determinations in parentheses. Where only two numbers are shown, there was not enough of one of the samples to permit the enzyme assay. Fractions D and E never accounted for more than 4% of the total protein recovered from the gradients, and when enough was obtained to permit ACC assay, the specific activities were about the same as those in fraction A or A'. Sources of enzyme activity

A CC specific activities in high-speed supernatants from the fractions

minced tissue screened tissue

1.80 _ 0.27 (4) 1.30 + 0.48 (4)

Fractions (1.0 M suc. ) light A medium B heavy C

0.86 + 0.15 (3) 1.59 + 0.18 (3) 0.34 _+0.35 (3)

Fractions (0.85 M suc. ) light A' medium B' heavy C'

0.44, 0.53 1.14 _+0.36 (3) 0.39 _+0.11 (3)

>

o

T.2

c

B

1.o

~ 0.8 ~

0.6

~® 0.4 u u

0.2

B

C

Fraction

Fig. 3. Comparison between distribution of acetyl-CoA carboxylase (ACC) and protein in the major fractions from the density gradients. In each experiment the amount of protein recovered in each fraction and the total activity of ACC recovered in each fraction were determined. Then in each experiment total values for all fractions were calculated and the individual values divided by the totals to obtain % enzyme recovered and % protein recovered in each fraction. Then for each fraction the % enzyme recovered was divided by the % protein recovered to obtain an ACC-to-protein recovery ratio. The means from 6 experiments are shown, with standard deviations. Solid bar, fraction A; open bar, fraction B; shaded bar, fraction C.

127 min/mg protein (Table II). If supernatants prepared from the least dense fractions, A and A ' , were compared to another, the material remaining suspended in 0.85 M sucrose (i.e. A ' ) had lower A C C specific activity than did the respective material collecting in 1.0 M sucrose (Fraction A). Since any myelin present would collect in this fraction, this result implied that vesicles of immature myelin might be a site of relatively low A C C activity, if any. There also was A C C activity in the fraction which sedimented through 1.2

.....

~

~

x9 ....

M sucrose during the second centrifugation step (Fractions C and C', Table II). W h e n ACC-to-protein recovery ratios were calculated from the combined data, the mean value for B was higher than those for A or C at P < 0.001 (Fig. 3). This showed that the high recovery of A C C in the medium density fractions was not merely the result of high protein recovery in those fractions. Because of the potential contamination by erythrocytes the fractions were not distinguished by C A

l[

Fig. 4. Samples of isolated fractions, after immunostaining for GC or CA. Several drops of sample were permitted to dry on each gelatin-coated slide. The slides were then incubated with the following series of reagents, all in phosphate-buffered saline (per liter, 0.2 g NaHEPO4, 1.15 g Na2HPO4-7H20 , 8 g NaCI)(PBS) 3% H202, 10 min; 30% normal s~,ine serum 15 rnj~a;anti,CA 1:20 o~¢ei-riigfitOr anti-GC 1:100 two hours, both in 1% normal swine serum: swine anti-rabbit IgG 1:20 30 min; ral~bit peroxidase-antiperoxidase 1:50 in 1% normal swine serum 30 min; 3,3'-diaminobenzidine (DAB) plus 0.01% H2028 min in the dark. Slides were washed with PBS between steps, except after the normal swine serum. The slides were dehydrated in graded ethanol solutions, followed by xylene, and mounted with Permount. The fractions and primary antisera are: a, fraction A, anti-ca; b, fraction A, anti GC; c, fraction B, anti-ca; d, fraction B, anti-GC; e, fraction C, anti-ca; f, fraction C, anti-GC; g, fraction D, anti-ca; h, fraction E, anti-GC. All the magnifications are the same, and scale bar = 5/zm.

assays but, rather, were examined by light microscopy. The fractions were first immunostained with antibodies against GC and CA, both of which are most concentrated in oligodendrocytes and myelin (reviewed in ref. 13). Some immunostaining was apparent: however, because the tissue was unfixed and was from very young animals, cytoplasmic C A was not heavily stained, nor was GC staining very intense (Fig. 4c-e). However, the light immunostaining of membrane-bound C A and of GC enabled visualization of the cells and clumps of membrane vesicles, and some even lighter staining, which appeared nonspecific, permitted visualization of a few surviving neurons and capillaries (e.g. Fig. 4f,g). Fixation destroyed the GC (not shown). The least dense fractions contained clumps of material probably consisting of immature myelin and other membrane fragments (Fig. 4a,b); the medium-density fractions, B and B', contained cells, often in clumps, many of which stained with anti-GC or anti-CA, and which, therefore, were probably oligodendrocytes or immature oligodendrocytes (e.g. arrowheads in Fig. 4c,d) and possibly some red blood cells (not shown); the

a

Fig. 5. Immunostaining of fractions for ACC. Samples of the fractions were stained as described in the legend to Fig. 4. The anti-ACC was incubated on the slides overnight at 1:500; in panel a normal rabbit serum was used in place of anti-ACC. The fractions shown are: a and c, fraction B; b, fraction D; d, fraction C. All the magnifications are the same, and scale bar = l(lum.

relatively dense fractions, ( and C', contained some of the putative oligodendrocytes (Fig. 4el, some capillaries (Fig. 4f), and some contracted erythrocytes (e.g. arrowhead in Fig. 4el. The lighter-stained, or unstained, cells in fractions B and C (e.g. arrow in Fig. 4c) probably were immature glial cells, judging from the pale nuclei observed in samples embedded in Epon and stained with toluidine blue (not shown). Fractions D and E contained little material, and only a few cells and/or clumps of debris could be found, on the slides (Fig. 4g,h). The large structures, (e.g. arrowhead in Fig. 4g) may represent a small population of neurons that were able to survive the salt solution in the absence of trypsin. The cells in fraction B, but, for the most part, not in A (not shown), C, D, or E (not shown) also immunostained with antibodies against A C C (Fig. 5). DISCUSSION The present values for A C C in forebrain fall within the range of values in the literature, and our values in brainstem are higher than those reported previously (Table I). The results of all the studies show decreases during development, and most of the differences in specific activities may be attributable to unavoidable inaccuracies in converting units per gram brain into units per mg supernatant protein and the use of whole brain and forebrain by different investigators. Early in this investigation we observed that a major source of potential inaccuracy in the assay was incomplete expulsion of unreacted Hi4CO3 as CO 2, implying that methanol had to be added and drying continued overnight. Incomplete removal of unreacted isotope can result either in artifactually low activities, due to high counts at to, or to artifactually high activities due to high counts at later time points. Thus, it is difficult to evaluate the differences among A C C activities reported in the literature. When performing fractionation of brain tissue, the preincubation in the presence of citrate and the elimination of a proteolytic step made it possible to retain significant A C C activity. While the fractions sedimented at different densities, and material was somewhat more spread-out on the gradient, than when established methods were used 9A2, the maximal specific activities and recovery of A C C were found in a fraction where immunocytochemical staining showed

129 a significant content of oligodendrocytes. This fraction, which showed the highest specific activity of A C C (Table II) and preferential recovery of A C C over protein (Fig. 3), sedimented at a lower density (i.e. above 1.2 M sucrose) than did the oligodendrocytes prepared by Snyder et al. 12 (i.e. below 1.4 M sucrose). The omission of trypsin was probably responsible for the difference in properties of the cells in that the cells prepared in our laboratory were clumped together, possibly due to the presence of undigested connections between oligodendrocytes and the beginnings of myelinated internodes along some of the axons. Even a low content of early turns of myelin could account for the small decrease in density, since the most dense fraction of isolated myelin sediments above 0.70 M sucrose 7. It should also be noted that in an earlier study, where a hexose-containing medium was used for cell isolation from rat brain 9, a fraction apparently comprising all the glial cell types collected above 1.35 M sucrose. In the neutral solutions containing salts ( - 300 mOs), which were used in the present study and that of Snyder et al. t2, most of the neurons and astrocytes in that fraction would have been lysed. Thus, the collection of oligodendrocyte-containing clumps above 1.2 M sucrose is not entirely inconsistent with the results of previous studies. In fact, judging by the intense carbonic anhydrase and galactocerebroside immunostaining and the small rounded cell shape, free of visible processes, oligodendrocytes contributed heavily to this cell population. While A C C was recovered preferentially and

showed significant specific activity in the oligodendrocyte-rich fraction, the A C C specific activity was not significantly higher in that fraction (B and B' in Table II) than in the screened homogenate. However, it is likely, because of the general lability and cytoplasmic location of this enzyme, that some A C C activity was lost during centrifugation on the density gradient, and it should be noted that the specific activities in fractions B and B' were much higher than those in the fractions sedimenting at higher or lower densities (Table II). The presence of some A C C activity in the fraction containing some putative neurons (legend to Table II and panel g of Fig. 4) is not surprising, since this enzyme would be required for lipid biosynthesis in that cell type, although at rates lower than those in myelinating oligodendrocytes. In the fraction (B or B') which had the highest A C C specific activity, the only detectable non-glial cell type was the red blood cell. When we prepared red blood cell lysates and used >250 pg protein per tube in A C C assays, no enzyme activity was detectable (our unpublished data). It is likely, therefore, that if it were possible to retain more A C C activity, the A C C specific activities in the cells would be higher than those reported here.

REFERENCES

York, 1972, pp. 3-11. 7 Matthieu, J.-M., Quarles, R.H., Brady, R.O. and Webster, H. deF., Variation of proteins, enzyme markers, and gangliosides in myelin subfractions, Biochim. Biophys, Acta, 329 (1973) 305-317. 8 Mead, J.F. and Dhopeshwarkar, G.A., Types of fatty acids in brain lipids, their derivation and function. In Lipids, Malnutrition and the Developing Brain, Ciba Foundation Symposium, Associated Scientific, New York, 1972, pp. 59-72. 9 Norton, W.T. and Poduslo, S.E., Neuronal soma and whole neuroglia of rat brain: a new isolation technique, Science, 167 (1970) 1144-1146. 10 Patel, M.S. and Owen, O.E., Lipogenesis from ketone bodies in rat brain, Biochem. J., 156 (1976) 603-607. 11 Pate!, M.S. and Tonkonow, B.L., Development of lipogenesis in rat brain cortex: the differential incorporation of glucose and acetate into brain lipids in vitro, J. Neurochem., 23 (1974) 309-313.

1 Ahmad, P.M., Gupta, S., Barden, R.E. and Ahmad, F., Inhibitory effects of sulfhydryl reagents on acetyl-CoA carboxylase from rat mammary gland, Biochim. Biophys. Acta, 789 (1984) 152-158. 2 Brady, R.O., Biosynthesis of fatty acids. II. Studies with enzymes obtained from brain, J. Biol. Chem., 235 (1960) 3099-3103. 3 Cheng, S.-C., CO 2 fixation in the nervous tissue, Int. Rev. Neurobiol., 14 (1971) 125-157. 4 Edmond, J., Ketone bodies as precursors of sterols and fatty acids in the developing rat, J. Biol. Chem., 249 (1974) 72-80. 5 Gross, I. and Warshaw, J.B., Fatty acid synthesis in developing brain, Biol. Neonate, 25 (1974) 365-375. 6 Inoue, H. and Lowenstein, J.M., Acetyl coenzyme A carboxylase from rat liver. In J.M. Lowenstein (Ed.), Methods in Enzymology, Vol. 35, Lipids, Part B, Academic, New

ACKNOWLEDGEMENTS This work was supported by US.P.H.S. Grant NS12890 and by Grant 1089 from the National Multiple Sclerosis Society.

13(/ 12 Snyder, D.S., Raine, C.S., Farooq, M. and Norton, W.T., The bulk isolation of oligodendroglia from whole rat forebrain: a new procedure using physiologic media, J. Neurochem., 34 (1980) 1614-1621. 13 Sternberger, N.H., Patterns of oligodendrocyte function seen by immunocytochemistry, Adv. Neurochem., 5 (1984) 125-173. 14 Wakil, S.J., Stoops, J.K. and Joshi, V.C., Fatty acid synthesis and its regulation, Annu. Rev. Biochem., 52 (1983)

537-579. 15 Yeh, Y.-Y., Streuli, V.L. and Paulus, Z., Ketone bodies serve as important precursors of brain lipids in the developing rat, Lipids, 12 (1977) 957-964. 16 Yeh, Y.-Y,, Ginsburg, J .R. and Tso, T.B., Changes in lipogenic capacity and activities of ketolytic and lipogenic enzymes in brain regions of developing rats, J. Neurochem., 40 (1983) 99-105.