358
Brain Research, 93 (1975) 358362 !(.3 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Evidence for presence of UDP-galactose:ceramide galactosyltransferase in rat myelin
ELVIRA COSTANTINO-CECCARINI AND KUNIHIKO SUZUKI Saul R. Korey Department of Neurology, Department of Neuroscience, and Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, 17. Y. 10461 (U.S.A.)
(Accepted April 29th, 1975)
UDP-galactose:ceramide galactosyltransferase (EC 2.4.1.62) catalyzes the last step of synthesis of galactosylceramide2,s,9 which is a major lipid component o f the myelin sheath. UDP-glucose:ceramide glucosyltransferase is an analogous enzyme involved in the synthesis of glucosylceramide which is the precursor for ganglioside synthesisl,lL Both enzymes are membrane-bound and generally considered to be localized in microsomes. However, a report by Neskovic et al. 1° suggested the presence o f galactosyltransferase in rat myelin. Since the question is important in relation to the formation and maintenance of the myelin sheath, we attempted to obtain more convincing evidence for possible synthesis of galactosylceramide within myelin. Brains from 20-22-day-old Sprague-Dawley rats were used throughout. Purified myelin was prepared by the method o f Norton and PoduslolL The standard subceltular fractionation was carried out according to Clendenon and Allen a. Microsomal subfractions were prepared by layering standard microsomes, suspended in 4 ml o f 0.32 M sucrose, on a discontinuous sucrose density gradient made up with 3 ml each of 1.3 M, 1.0 M, 0.75 M, and 0.5 M. Centrifugation was at 80,000 × g for 120 min in a Spinco SW-27 rotor. The fractions at the interfaces and the pellet were separately collected, diluted with cold water and centrifuged at 105,000 × g for 60 min. Details of the assays for the two glycosyltransferases have been described 5,s,9. The galactosyltransferase was assayed with 1 mg/tube o f additional ethanolamine phospholipid 5. Protein determination was by the method o f Lowry et al. n. When subcellular localization of the two glycosyttransferases was examined by the conventional fractionation procedure, 75-80 ~ of the recovered activity of glucosyltransferase was present in the microsomal fraction with a total recovery o f 77 ~. In contrast, only 40-45 ~ of total galactosyltransferase activity could be recovered in microsomes, although the overall recovery was 95 ~. Subfractionation of the crude mitochondrial fraction showed that myelin-rich and synaptosome-rich fractions contained galactosyltransferase with relative specific activities o f 3.2 and 3.8, respectively, over the starting homogenate. The relative specific activity for this enzyme in microsomes was 2.3. These findings, except for the relatively high activity in the synapto-
359 TABLE I RAT BRAIN U D P - G A L A C T O S E I C E R A M I D E GALACTOSYLTRANSEERASE 1N MICROSOMAL AND MYELIN FRACTIONS
U DP-Glucose:ceramide glucosyltransferase was active in all microsomal fractions (0.86 !: 0.14 nmoles/h/mg protein) but was undetectable in all myelin fractions.
Experiment
Galactosyltransferase Microsomes Myelin (nmole/h/mg protein)
I 2 3 4 5
2.31 2.03 1.7o 1.30 2.80
0.97 0.93 1.81 1.40 1.30
Average ± S.D.
2.03 ~ 0.57
1.28 4 0.36
some-rich fraction, are similar to those of Neskovic et al. lo a n d suggest the possible presence of galactosyltransferase in myelin. The highly purified myelin fraction consistently c o n t a i n e d substantial activities of galactosyltransferase with specific activity similar to a n d at least 40 ~o of t h a t of microsomes while n o activity of glucosyltransferase could be detected (Table I). A series of experiments was therefore initiated in a n a t t e m p t to exclude n o n - m y e l i n i m p u r i t y as the source of galactosyltransferase in the purified myelin fraction. A mixing experiment was carried out in order to assess the degree of m i c r o s o m a l
TABLE It MIXING EXPERIMENT
Whole homogenate from 3 brains were divided into two equal parts. Myelin was isolated from one part directly. Microsomes obtained from 4 brains were added to the second part, the mixture rehomogenized, and then myelin was isolated.
Enzyme source
Microsomes Myelin (direct) Myelin (mixed)
Total protein (mg)
136 6.33 6.50
Galactosyltrans~erase
GlucosyltransJerase
Specific activity (nmole/h/mg protein)
Total activity (nmole/h)
Specific activity (nmole/h/mg protein)
Total activity (nmole/h)
2.31 0.97 1.09
314 6.14 7.09
1.0 n.d.* n.d.*
136
Calculated contamination from added microsomes: of added microsome 0.1 ~ 0.3 % of myelin fraction 3 ~ 13 * n.d. = not detectable.
N
360 TABLE 111 RAT BRAIN CERAMIDE (.JLYCOSYLrRANSFERASES IN SUBMICROSOMAL FRACTIONS
Fractions* *
Protein
Galactosyltransferase
GlucosvltransJbrase
Ratio
(rag~brain)
(Gal)
(Glc)
Glc/Gul
Specific activity (nmole/h/ mg protein)
Total activiO' (nmole/h/ brain)
Specific activity (nmote/h/ mg protein
Total activity (nmole/h/ brain)
Total microsomes 30.7 0.5 M 4.23 0.75 M 6.35 1.0 M 6.01 1.3 M 3.90 pellet 1.30
2.25 1.93 3.24 2.38 1.47 0.91
69.1 8.16 20.6 14.3 5.73 1.18
0.98 0.92 1.82 0.74 0.06 n.d.*
30.1 3.89 11.6 4.45 0.23
Recovery
....
72 ",,
71 oo
--.
0.44 0.48 0.56 0.31 0.04 0
67 %
• n.d. - not detectable. • * Submicrosomal fractions are designated by molarity of sucrose on which respective fractions float.
contamination (Table II). The yield and the activity of galactosyltransferase in the two myelin fractions, one isolated directly from whole brain homogenate and the other isolated from a mixture o f whole brain homogenate and additional excess microsomes, were essentially identical within experimental errors. No activity of glucosyltransferase was detected in either of the myelin fractions, while it was active in microsomes. To rule out preferential inactivation ofglucosyltransferase during myelin isolation, the microsomal fraction was subjected to additional procedures designed to simulate the conditions of the myelin isolation procedure. The standard microsomal fraction was suspended in distilled water and centrifuged at 105,000 × g for 60 min. Thisprocedure was repeated three times. There was no loss of activity of either of the gtycosyltransferases, and the activity ratio of glucosyl- to galactosyltransferases, before and after the osmotic shock and washing procedure, was 0.84 and 0.81, respectively. These experiments excluded the possibility of contamination by the general population of microsomes as the source o f the galactosyltransferase activity in our myelin fraction. The possibility still remained that a specific microsomal subfraction, highly enriched in galactosyltransferase but containing no glucosyltransferase, may be present in our myelin preparation as a contaminant. Therefore, microsomal subfractions, arbitrarily separated by their density, were examined for activities of the two glycosyltransferases (Table Ill). Recoveries of protein and of activities of the glycosyltransferases from the 5 subfractions were similar. The 3 lighter fractions contained 87 % of the recovered activit~ of galactosyltransferase and 99 % of the recovered activity of glucosyltransferase. In these lighter subfractions, the activity ratio of glucosyltransferase to gatactosyltransferase ranged from 0.31 to 0.56, while the ratio in the total microsomes was 0.44. The subfraction floating on 0.75 M sucrose had the highest specific activities of both glycosyltransferases. On the other hand, the 2 heavy frac-
361 tions, which contained 24 ~ of total recovered protein, showed substantial activities of galactosyltransferase but essentially no glucosyltransferase. Therefore, these two heavy microsomal subfractions resembled the purified myelin fraction with respect to the specific activities and distribution of the two glycosyltransferases. In this respect, these heavy submicrosomal fractions could be a possible contaminant to account for the galactosyltransferase activity in our myelin fraction. However, the densities of these subfractions are much higher than that of myelin. The 1.3 M subfraction consisted mostly of heterogenous membrane fragments with fairly abundant smooth vesicular structures and ribosomes when examined under the electron microscope. No myelin, identifiable as such by its ultrastructure, could be detected. Since the specific activity of galactosyltransferase was similar in the purified myelin fraction and in these heavy submicrosomal fractions, the entire myelin fraction would have to consist of these submicrosomal fractions to account for the galactosyltransferase activity of the myelin fraction. In view of the above ultrastructure, this is clearly not the case. Our findings are similar to those of Neskovic e t al. 1° in several respects. Specific activity of galactosyltransferase in their 'purified' myelin was 60 ~ of that of microsomes. On the other hand, specific activity of glucosyltransferase was 15 ~ of that in microsomes. They also reported relative enrichment of galactosyltransferase in heavier submicrosomal fractions, and glucosyltransferase in lighter subfractions. Their results, however, were less clear-cut than ours. We believe that this resulted from the less pure nature of their 'purified' myelin. For example, the residual activity of the glucosyltransferase in their myelin fraction was probably due to microsomal contamination. Absence of recovery data at various fractionation steps added ambiguity in interpretation of their data. Our present observations provide firmer evidence that highly purified myelin contains UDP-galactose:ceramide galactosyltransferase but not UDP-glucose:ceramide glucosyltransferase. We must be aware that our results still do not conclusively prove the presence of galactosyltransferase in the myelin sheath. A very specific small subcellular fraction with a very high specific activity of the enzyme can still be present in the myelin fraction. The possibility includes the axonal membrane 7 or a specific subfraction of the heavy submicrosomal fractions. However, these remaining possibilities seem unlikely because, whatever the contaminating fraction, it must contain galactosyltransferase at a specific activity at least 10 times that of microsomes, or 20-30 times that of whole homogenate if we assume 5 ~ impurity in the myelin fraction. It seems therefore reasonable to conclude tentatively that UDP-galactose :ceramide galactosyltransferase is present in the myelin sheath. At present there is no evidence that the enzyme in the myelin sheath is different from that localized in microsomes. Since galactosylceramide is the major and characteristic lipid of myelin, the finding of galactosyltransferase in myelin is potentially significant with respect to its metabolism. The total activity present in the myelin fraction is relatively minor, at least at the stage of brain development studied. It constituted approximately 5-10 ~ of the total activity of the enzyme in the whole brain. Whether this enzyme participates in galactosylation of some of galactosylceramide in the process of myelination or
362 w h e t h e r its role is for the m a i n t e n a n c e o f the myelin sheath after initial myelination is largely c o m p l e t e d r e m a i n s for future studies. W e t h a n k Dr. K i n u k o Suzuki, D e p a r t m e n t o f P a t h o l o g y , who examined the u l t r a s t r u c t u r e o f m i c r o s o m a l subfractions. The results o f this study were presented at the 6th A n n u a l M e e t i n g o f the A m e r i , can Society for N e u r o c h e m i s t r y held in M e x i c o City, M a r c h 10-14, 1975, a n d published in an a b s t r a c t form 4. This investigation was s u p p o r t e d by R e s e a r c h G r a n t s NS-10885, NS-03356 a n d HD-01799 f r o m the U n i t e d States Public H e a l t h Service, a n d by G r a n t 982-A-4 f r o m the N a t i o n a l M u l t i p l e Sclerosis Society.
1 BASU,S., KAUFMAN,B., ANO ROSEMAN,S., Enzymatic synthesis ofceramide glucose and ceramide lactose by glycosyltransferases from embryonic chicken brain, J. biol. Chem., 243 (1968) 58025804. 2 BASU,S., SCHULTZ,A. M., BASU,M., ANOROSEMAN,S., Enzymatic synthesis of galactocerebrosides by a galactosyltransferase from embryonic chicken brain, J. biol. Chem., 246 (1971) 4272-4279. 3 CLENOENON,N. R. ANO ALLEN, N., Assay of subcellular localization of the arylsulfatases in rat brain, Z Neurochem., 17 (1970) 865-879. 4 COSTANTINO-CECCARINI.E., AND SUZUKI, K., UDP-Galactose:ceramide galactosyltransferase in rat myelin. Trans. Amer. Soc. Neurochem.. 6 (1975) 212. 5 COSTANTINO-CEccARINL E., AND SUZUKI, K., Effect of exogenous lipids on membrane-bound ceramide glycosyltransferases of rat brain, Arch. Biochem., 167 (1975) 646-654. 6 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J., Protein measuremertt with the Folin phenol reagent, d. biol. Chem., 193 (1951) 265-275. 7 MClLWAIN. D. L.. Localization of the acetylcholinesterase-containing membranes in purified myelin fractions, Brain Research, 69 (1974) 182-187. 8 MORELL, P., COSTANTINO°CECCARINI, E., AND RADIN, N. S., The biosynthesis by brain m~crosomes of cerebroside containing nonhydroxy fatty acids, Arch. Biochem., 141 (1970) 738-748. 9 MORELL, P.. AND RADIN. N. S.. Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid, Biochemistry, 8 (1969) 506--512. 10 NESKOVIC,N. M., SARLIEVE,L. L., AND MANDEL,P., Subcellular and submicrosomal distribution of glycolipid-synthesizingtransferases in young rat brain, J. Neurochem., 20 (1973) 1419-1430. 11 NORTON, W. T., AND PODUSLO. S. E., Myelination in rat brain: method of myelin isolation. J. Neurochem., 21 (1973) 749-757. 12 SVENNERI-IOLM,L., Gangliosides. In A. LAJTHA (Ed.), Handbook of Neurochemistry, VoL 3, Plenum Press, New York, 1970, pp. 425-452.
Brain Research, 93 (1975) 358362 !(.3 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Evidence for presence of UDP-galactose:ceramide galactosyltransferase in rat myelin
ELVIRA COSTANTINO-CECCARINI AND KUNIHIKO SUZUKI Saul R. Korey Department of Neurology, Department of Neuroscience, and Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, 17. Y. 10461 (U.S.A.)
(Accepted April 29th, 1975)
UDP-galactose:ceramide galactosyltransferase (EC 2.4.1.62) catalyzes the last step of synthesis of galactosylceramide2,s,9 which is a major lipid component o f the myelin sheath. UDP-glucose:ceramide glucosyltransferase is an analogous enzyme involved in the synthesis of glucosylceramide which is the precursor for ganglioside synthesisl,lL Both enzymes are membrane-bound and generally considered to be localized in microsomes. However, a report by Neskovic et al. 1° suggested the presence o f galactosyltransferase in rat myelin. Since the question is important in relation to the formation and maintenance of the myelin sheath, we attempted to obtain more convincing evidence for possible synthesis of galactosylceramide within myelin. Brains from 20-22-day-old Sprague-Dawley rats were used throughout. Purified myelin was prepared by the method o f Norton and PoduslolL The standard subceltular fractionation was carried out according to Clendenon and Allen a. Microsomal subfractions were prepared by layering standard microsomes, suspended in 4 ml o f 0.32 M sucrose, on a discontinuous sucrose density gradient made up with 3 ml each of 1.3 M, 1.0 M, 0.75 M, and 0.5 M. Centrifugation was at 80,000 × g for 120 min in a Spinco SW-27 rotor. The fractions at the interfaces and the pellet were separately collected, diluted with cold water and centrifuged at 105,000 × g for 60 min. Details of the assays for the two glycosyltransferases have been described 5,s,9. The galactosyltransferase was assayed with 1 mg/tube o f additional ethanolamine phospholipid 5. Protein determination was by the method o f Lowry et al. n. When subcellular localization of the two glycosyttransferases was examined by the conventional fractionation procedure, 75-80 ~ of the recovered activity of glucosyltransferase was present in the microsomal fraction with a total recovery o f 77 ~. In contrast, only 40-45 ~ of total galactosyltransferase activity could be recovered in microsomes, although the overall recovery was 95 ~. Subfractionation of the crude mitochondrial fraction showed that myelin-rich and synaptosome-rich fractions contained galactosyltransferase with relative specific activities o f 3.2 and 3.8, respectively, over the starting homogenate. The relative specific activity for this enzyme in microsomes was 2.3. These findings, except for the relatively high activity in the synapto-
359 TABLE I RAT BRAIN U D P - G A L A C T O S E I C E R A M I D E GALACTOSYLTRANSEERASE 1N MICROSOMAL AND MYELIN FRACTIONS
U DP-Glucose:ceramide glucosyltransferase was active in all microsomal fractions (0.86 !: 0.14 nmoles/h/mg protein) but was undetectable in all myelin fractions.
Experiment
Galactosyltransferase Microsomes Myelin (nmole/h/mg protein)
I 2 3 4 5
2.31 2.03 1.7o 1.30 2.80
0.97 0.93 1.81 1.40 1.30
Average ± S.D.
2.03 ~ 0.57
1.28 4 0.36
some-rich fraction, are similar to those of Neskovic et al. lo a n d suggest the possible presence of galactosyltransferase in myelin. The highly purified myelin fraction consistently c o n t a i n e d substantial activities of galactosyltransferase with specific activity similar to a n d at least 40 ~o of t h a t of microsomes while n o activity of glucosyltransferase could be detected (Table I). A series of experiments was therefore initiated in a n a t t e m p t to exclude n o n - m y e l i n i m p u r i t y as the source of galactosyltransferase in the purified myelin fraction. A mixing experiment was carried out in order to assess the degree of m i c r o s o m a l
TABLE It MIXING EXPERIMENT
Whole homogenate from 3 brains were divided into two equal parts. Myelin was isolated from one part directly. Microsomes obtained from 4 brains were added to the second part, the mixture rehomogenized, and then myelin was isolated.
Enzyme source
Microsomes Myelin (direct) Myelin (mixed)
Total protein (mg)
136 6.33 6.50
Galactosyltrans~erase
GlucosyltransJerase
Specific activity (nmole/h/mg protein)
Total activity (nmole/h)
Specific activity (nmole/h/mg protein)
Total activity (nmole/h)
2.31 0.97 1.09
314 6.14 7.09
1.0 n.d.* n.d.*
136
Calculated contamination from added microsomes: of added microsome 0.1 ~ 0.3 % of myelin fraction 3 ~ 13 * n.d. = not detectable.
N
360 TABLE 111 RAT BRAIN CERAMIDE (.JLYCOSYLrRANSFERASES IN SUBMICROSOMAL FRACTIONS
Fractions* *
Protein
Galactosyltransferase
GlucosvltransJbrase
Ratio
(rag~brain)
(Gal)
(Glc)
Glc/Gul
Specific activity (nmole/h/ mg protein)
Total activiO' (nmole/h/ brain)
Specific activity (nmote/h/ mg protein
Total activity (nmole/h/ brain)
Total microsomes 30.7 0.5 M 4.23 0.75 M 6.35 1.0 M 6.01 1.3 M 3.90 pellet 1.30
2.25 1.93 3.24 2.38 1.47 0.91
69.1 8.16 20.6 14.3 5.73 1.18
0.98 0.92 1.82 0.74 0.06 n.d.*
30.1 3.89 11.6 4.45 0.23
Recovery
....
72 ",,
71 oo
--.
0.44 0.48 0.56 0.31 0.04 0
67 %
• n.d. - not detectable. • * Submicrosomal fractions are designated by molarity of sucrose on which respective fractions float.
contamination (Table II). The yield and the activity of galactosyltransferase in the two myelin fractions, one isolated directly from whole brain homogenate and the other isolated from a mixture o f whole brain homogenate and additional excess microsomes, were essentially identical within experimental errors. No activity of glucosyltransferase was detected in either of the myelin fractions, while it was active in microsomes. To rule out preferential inactivation ofglucosyltransferase during myelin isolation, the microsomal fraction was subjected to additional procedures designed to simulate the conditions of the myelin isolation procedure. The standard microsomal fraction was suspended in distilled water and centrifuged at 105,000 × g for 60 min. Thisprocedure was repeated three times. There was no loss of activity of either of the gtycosyltransferases, and the activity ratio of glucosyl- to galactosyltransferases, before and after the osmotic shock and washing procedure, was 0.84 and 0.81, respectively. These experiments excluded the possibility of contamination by the general population of microsomes as the source o f the galactosyltransferase activity in our myelin fraction. The possibility still remained that a specific microsomal subfraction, highly enriched in galactosyltransferase but containing no glucosyltransferase, may be present in our myelin preparation as a contaminant. Therefore, microsomal subfractions, arbitrarily separated by their density, were examined for activities of the two glycosyltransferases (Table Ill). Recoveries of protein and of activities of the glycosyltransferases from the 5 subfractions were similar. The 3 lighter fractions contained 87 % of the recovered activit~ of galactosyltransferase and 99 % of the recovered activity of glucosyltransferase. In these lighter subfractions, the activity ratio of glucosyltransferase to gatactosyltransferase ranged from 0.31 to 0.56, while the ratio in the total microsomes was 0.44. The subfraction floating on 0.75 M sucrose had the highest specific activities of both glycosyltransferases. On the other hand, the 2 heavy frac-
361 tions, which contained 24 ~ of total recovered protein, showed substantial activities of galactosyltransferase but essentially no glucosyltransferase. Therefore, these two heavy microsomal subfractions resembled the purified myelin fraction with respect to the specific activities and distribution of the two glycosyltransferases. In this respect, these heavy submicrosomal fractions could be a possible contaminant to account for the galactosyltransferase activity in our myelin fraction. However, the densities of these subfractions are much higher than that of myelin. The 1.3 M subfraction consisted mostly of heterogenous membrane fragments with fairly abundant smooth vesicular structures and ribosomes when examined under the electron microscope. No myelin, identifiable as such by its ultrastructure, could be detected. Since the specific activity of galactosyltransferase was similar in the purified myelin fraction and in these heavy submicrosomal fractions, the entire myelin fraction would have to consist of these submicrosomal fractions to account for the galactosyltransferase activity of the myelin fraction. In view of the above ultrastructure, this is clearly not the case. Our findings are similar to those of Neskovic e t al. 1° in several respects. Specific activity of galactosyltransferase in their 'purified' myelin was 60 ~ of that of microsomes. On the other hand, specific activity of glucosyltransferase was 15 ~ of that in microsomes. They also reported relative enrichment of galactosyltransferase in heavier submicrosomal fractions, and glucosyltransferase in lighter subfractions. Their results, however, were less clear-cut than ours. We believe that this resulted from the less pure nature of their 'purified' myelin. For example, the residual activity of the glucosyltransferase in their myelin fraction was probably due to microsomal contamination. Absence of recovery data at various fractionation steps added ambiguity in interpretation of their data. Our present observations provide firmer evidence that highly purified myelin contains UDP-galactose:ceramide galactosyltransferase but not UDP-glucose:ceramide glucosyltransferase. We must be aware that our results still do not conclusively prove the presence of galactosyltransferase in the myelin sheath. A very specific small subcellular fraction with a very high specific activity of the enzyme can still be present in the myelin fraction. The possibility includes the axonal membrane 7 or a specific subfraction of the heavy submicrosomal fractions. However, these remaining possibilities seem unlikely because, whatever the contaminating fraction, it must contain galactosyltransferase at a specific activity at least 10 times that of microsomes, or 20-30 times that of whole homogenate if we assume 5 ~ impurity in the myelin fraction. It seems therefore reasonable to conclude tentatively that UDP-galactose :ceramide galactosyltransferase is present in the myelin sheath. At present there is no evidence that the enzyme in the myelin sheath is different from that localized in microsomes. Since galactosylceramide is the major and characteristic lipid of myelin, the finding of galactosyltransferase in myelin is potentially significant with respect to its metabolism. The total activity present in the myelin fraction is relatively minor, at least at the stage of brain development studied. It constituted approximately 5-10 ~ of the total activity of the enzyme in the whole brain. Whether this enzyme participates in galactosylation of some of galactosylceramide in the process of myelination or
362 w h e t h e r its role is for the m a i n t e n a n c e o f the myelin sheath after initial myelination is largely c o m p l e t e d r e m a i n s for future studies. W e t h a n k Dr. K i n u k o Suzuki, D e p a r t m e n t o f P a t h o l o g y , who examined the u l t r a s t r u c t u r e o f m i c r o s o m a l subfractions. The results o f this study were presented at the 6th A n n u a l M e e t i n g o f the A m e r i , can Society for N e u r o c h e m i s t r y held in M e x i c o City, M a r c h 10-14, 1975, a n d published in an a b s t r a c t form 4. This investigation was s u p p o r t e d by R e s e a r c h G r a n t s NS-10885, NS-03356 a n d HD-01799 f r o m the U n i t e d States Public H e a l t h Service, a n d by G r a n t 982-A-4 f r o m the N a t i o n a l M u l t i p l e Sclerosis Society.
1 BASU,S., KAUFMAN,B., ANO ROSEMAN,S., Enzymatic synthesis ofceramide glucose and ceramide lactose by glycosyltransferases from embryonic chicken brain, J. biol. Chem., 243 (1968) 58025804. 2 BASU,S., SCHULTZ,A. M., BASU,M., ANOROSEMAN,S., Enzymatic synthesis of galactocerebrosides by a galactosyltransferase from embryonic chicken brain, J. biol. Chem., 246 (1971) 4272-4279. 3 CLENOENON,N. R. ANO ALLEN, N., Assay of subcellular localization of the arylsulfatases in rat brain, Z Neurochem., 17 (1970) 865-879. 4 COSTANTINO-CECCARINI.E., AND SUZUKI, K., UDP-Galactose:ceramide galactosyltransferase in rat myelin. Trans. Amer. Soc. Neurochem.. 6 (1975) 212. 5 COSTANTINO-CEccARINL E., AND SUZUKI, K., Effect of exogenous lipids on membrane-bound ceramide glycosyltransferases of rat brain, Arch. Biochem., 167 (1975) 646-654. 6 LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J., Protein measuremertt with the Folin phenol reagent, d. biol. Chem., 193 (1951) 265-275. 7 MClLWAIN. D. L.. Localization of the acetylcholinesterase-containing membranes in purified myelin fractions, Brain Research, 69 (1974) 182-187. 8 MORELL, P., COSTANTINO°CECCARINI, E., AND RADIN, N. S., The biosynthesis by brain m~crosomes of cerebroside containing nonhydroxy fatty acids, Arch. Biochem., 141 (1970) 738-748. 9 MORELL, P.. AND RADIN. N. S.. Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid, Biochemistry, 8 (1969) 506--512. 10 NESKOVIC,N. M., SARLIEVE,L. L., AND MANDEL,P., Subcellular and submicrosomal distribution of glycolipid-synthesizingtransferases in young rat brain, J. Neurochem., 20 (1973) 1419-1430. 11 NORTON, W. T., AND PODUSLO. S. E., Myelination in rat brain: method of myelin isolation. J. Neurochem., 21 (1973) 749-757. 12 SVENNERI-IOLM,L., Gangliosides. In A. LAJTHA (Ed.), Handbook of Neurochemistry, VoL 3, Plenum Press, New York, 1970, pp. 425-452.
