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Proc. Natl. Acad. Sci. USA Vol. 76, No. 9, pp. 4270-4274, September 1979 Differential activities of glycolipid in Tay- Sachs disease: Studies in cultured cells from cerebrum (/blood group-related /galactosyltransferases//fucosyltransferases) MANJU BASU*, KATHLEEN A. PRESPER*, SUBHASH BASU*, LINDA M. HOFFMANt, AND STEVEN E. BROOKSt *Department of Chemistry, Biochemistry and Biophysics Program, University of Notre Dame, Notre Dame, Indiana 46556; and tNeuroscience Center of the Kingsbrook Jewish Medical Center and the Downstate Medical Center of the State University of New York, Brooklyn, New York 11203 Communicated by Edwin T. Mertz, May 29, 1979

ABSTRACT Four different glycolipid: Table 1. The present report is concerned with the biosynthesis activities involved in the biosynthesis in vitro of gangliosides of sialylneolactotetraosylceramide (AcNeu-nLcOse4Cer) (15, and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial culture derived from 16) in TSD transformed cells and the lack of synthesis of either the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD GM1 (11, 17) or blood group-active glyco- cultured brain cells contained little activity of either UDP- HI (17, 18). Gal:GM2 (81-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GMla from GM2 (Tay-Sachs) gan- MATERIALS AND METHODS glioside, or GDP-Fuc:nLcOse4Cer (al-2)fucosyltransferase (FucT-2; EC 2.4.1.89), which catalyzes the formation of HI gly- Cell Culture. TSD cell cultures were established at Kings- colipid from nLcOse4Cer. These cells contained a potent in- brook Jewish Medical Center (12). The cells were passaged hibitor of the second reaction (catalyzed by a Golgi-rich mem- serially as diploid strains from the cerebrum of a 20-week-old brane fraction from bovine spleen), whereas no inhibition of TSD fetus. Cultures established from TSD brain displayed the first reaction (catalyzed by a membrane fraction from 14- typical glia-like morphology. Cultures were transformed with day-old embryonic chicken brain) was observed. The activity the DNA virus SV40 in order to establish a permanent cell line of UDP-al:LcOse3Cer (f1-4)galactosyltransferase (GaIT4; EC 2.4.1.86) was 30- to 80fold higher than the activity of GalT-3. The (Fig. la). Cultures were grown in 250-ml Falcon plastic flasks presence of CMP-AcNeu:nLcOse4Cer activity containing 15 ml of Eagle's minimal essential medium sup- and the absence of either GaIT-3 or FucT-2 suggested a probable plemented with antibiotics (penicillin/streptomycin/Fungi- pathway for the synthesis of sialylneolactotetraosylceramide zone; per ml, 100 units/100,g/1.25 ug), 10% fetal bovine [GM1b(GlcNAc)] in addition to a specific blockage of GMla serum, and nonessential amino acids. The cells were incubated ganglioside synthesis from GM2 in these TSD transformed in a humidified atmosphere of 95% air/5% CO2 and harvested cells. with Hanks' balanced salt solution (Ca2+- and Mg2+-free) Tay-Sachs disease (TSD) is one of several ganglioside storage containing 0.1% EDTA and 0.1% trypsin. A strain of these cells diseases. It is classified as GM2-gangliosidosis type I because of showing a reduced anchorage dependence (Fig. lb) was isolated its neuronal accumulation of GM2 ganglioside (1-3) and its according to the method described by Risser and Pollack (19). asialo derivative (4), gangliotriaosylceramide (GgOsesCer). The cells were washed three times with phosphate-buffered Since the publication by Klenk in 1939, it has been reported saline and centrifuged; the packed cells were homogenized in repeatedly that TSD is a clinically (5), morphologically (6, 7), 0.32 M sucrose containing 0.1% 2-mercaptoethanol and the and genetically (8) well-defined entity, but its exact biochemical homogenate was used as source. cause is evidently very complex. It has been observed that Materials. UDP-['4C] (274 mCi/mmol), CMP- cultured skin fibroblast cells from TSD patients lack 3-D-hex- [4-'4C]AcNeu (1.68 mCi/mmol), and GDP-L-[14C]fucose (174 osaminidase A (9), but these cultured cells do not accumulate mCi/mmol) were purchased from New England Nuclear (1 GM2 ganglioside. In addition to the absence of lysosomal hex- Ci = 3.7 X 1010 becquerels). Unlabeled UDP-galactose was osaminidase A (10), the accumulation of GM2 could be the purchased from Sigma. Unlabeled CMP-AcNeu and GDP-L- result of a severe deficiency of a synthetic enzyme such as fucose were prepared according to the methods of Kean and UDP-galactose:GM2 (f1-3)galactosyltransferase (11), which Roseman (20) and Schachter et al. (21), respectively. The blood catalyzes the synthesis of GM1. group B-active pentaglycosylceramide Gal(al-3)Gal(f31-4)- In an attempt to study TSD in cultured cells, Hoffman et al. GlcNAc(f1-3)Gal(31-4)Glc-Cer (nLcOse5Cer) was isolated (12) have investigated the gangliosides in a glial cell strain that from rabbit (22) and bovine (23, 24) erythrocytes. Both neo- was derived from the cerebellum of a TSD fetus. They have lactotetraosylceramide (nLcOse4Cer) [Gal(/31-4)GlcNAc(31- shown that there is a 5-fold increase in GM2 content in TSD cer- Abbreviations: TSD, Tay-Sachs disease; SV40, simian virus 40; ECBM, cerebellar cells compared with cultures derived from the embryonic chicken brain membrane; BSGM, bovine spleen Golgi-rich ebellum of a normal age-matched control. Retently Schneck membrane; LacCer, ; LcOse3Cer, lactotriaosylcer- and his coworkers (13) have established a permanent line of glial amide [GlcNAc(f1-3)Gal(f31-4)Glc-Cer]; nLcOse4Cer, neolacto- cells from fetal TSD cerebrum for the study of this disease. In tetraosylceramide [Gal(fll-4)GlcNAc(j1-3)Gal(f31-4)Glc-Cerl; addition to GM3 and GM2 gangliosides, Hoffman et al. (14) nLcOse5Cer, neolactopentaosylceramide [Gal(a1-3)Gal(f1-4)- have found a high level of N-acetylglucosamine-containing GlcNAc(f1-3)Gal(11-4)Glc-CerI; GgOse4Cer, gangliotetraosylcera- in these simian virus 40 TSD mide [Gal(l31-3)GalNAc(f3l-4)Gal(f3l-4)Glc-Cerl; GM3, hlematoside ganglioside (SV40)-transformed [AcNeu(a2-3)Gal(f31-4)Glc-Cer]; GM2, Tay-Sachs ganglioside [Gal- cells. This led us to investigate the four key reactions shown in NAc(f31-4)Gal(3-2 AcNeu)(31-4)Glc-Cer]; GalT-3, UDP-galac- tose:GM2 (31-3)galactosyltransferase (EC 2.4.1.62); GalT-4, UDP- The publication costs of this article were defrayed in part by page galactose:nLcOse4Cer (31-4)galactosyltransferase (EC 2.4.1.86); charge payment. This article must therefore be hereby marked "ad- FucT-2, GDP-fucose:nLcOse4Cer (al-2)fucosyltransferase (EC vertisement" in accordance with 18 U. S. C. §1734 solely to indicate 2.4.1.89); SAT-3, CMP-AcNeu:nLcOse4Cer (a2-3)sialyltransferase. this fact. abbreviations are according to ref. 38. 4270 Downloaded by guest on October 2, 2021 Biochemistry: Basu et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4271 Table 1. Enzymatic reactions studied in TSD cultures Activity Donor Acceptor Product (p1-3)Galactosyltransferase UDP-[14C]Gal GalNAc-Gal-Glc-Cer (GM2) GM1a (GalT-3; EC 2.4.1.62) AcNeu (f1-4)Galactosyltransferase UDP-[14C]Gal GlcNAc-Gal-Glc-Cer nLcOse4Cer (GalT-4; EC 2.4.1.86) (LcOse3Cer) (a2-3)Sialyltransferase (SAT-3) CMP-[14C]AcNeu Gal-GlcNAc-Gal-Glc-Cer GMlb(GlcNAc) (nLcOse4Cer) (al -2)Fucosyltransferase GDP-[14C]Fuc nLcOse4Cer HI (FucT-2; EC 2.4.1.89) (al-3)Galactosyltransferase (EC UDP-[14C]Gal nLcOse4Cer nLcOse5Cer (B) 2.4.1.87)

3)Gal(31-4)Glc-Cerj and lactotriaosylceramide (LcOse3Cer) were interpreted according to Bjorndal et al. (28). Fertilized [GlcNAc(31-3)Gal(31-4)Glc-Cer] were prepared by sequential eggs were obtained from Rose Hatchery (South Bend, IN). removal of terminal galactose units with purified fig a-galac- Purification of Membrane-Bound Glycosyltransferases. tosidase (25) and a combination of fig a-galactosidase and A Golgi-rich membrane fraction was prepared from fresh bo- papaya 3-galactosidase (16, 26), respectively. GM2 ganglioside vine spleen according to a published method (18) and was isolated from TSD brains according to a modification of contained high activity of GDP-L-fucose:nLcOse4Cer (al- the method of Svennerholm (2, 27). The purified glyco- 2)fucosyltransferase (FucT-2). The P3 membrane fraction were analyzed by gas/liquid chromatography which contained high UDP-galactose:GM2 (31-3)galactosyl- and gas chromatography/mass spectrometry. Mass spectral data (GalT-3) activity was prepared (29) from 14-day-old embryonic chicken brain. Glycolipid:Glycosyltransferase Assays. Complete incu- bation mixtures for the galactosyltransferase assay contained the following components (in Amol) in final volumes of 0.1 ml: acceptor glycosphingolipids, 0.05; Triton CF-54/Tween 80 (2:1), 0.3 mg; MnCl2, 0.25; sodium cacodylate/HCl buffer (pH 7.2), 10.0; UDP-[14C]galactose, 0.04 (1.85 X 106 cpm/,umol), and enzyme fractions consisting of homogenates of TSD cells, 0.15-0.3 mg, or membranes from embryonic chicken brain and bovine spleen, 0.14-0.2 mg of protein [estimated by the method of Lowry et al. (30)]. The mixtures were incubated for 2 hr at 37°C, and the reaction was stopped by the addition of 2.5 ,umol of EDTA and 10ll of chloroform/methanol (2:1, vol/vol). The whole liquid content and a 100-Al chloroform/methanol (2:1) wash of the tube were spotted on Whatman 3MM paper and assayed by a double chromatographic method described pre- viously (16, 26, 31). The radioactivities of appropriate areas of each chromatogram were determined quantitatively by liquid scintillation techniques with a Beckman scintillation counter (model LS-3133T). Incubation conditions for glycolipid sialyltransferase were the same as described above, except that the incubation mixture contained the following (in ,umol): cacodylate/HCl buffer (pH

Table 2. UDP-Gal:glycolipid galactosyltransferase activities in TSD transformed cerebrum cells ['4C]Galactose incorporated, Acceptor pmol/mg protein per 2 hr (0.5 mM) Linkage TSD-agar TSD ECBM (P3) 2-OH-Cer 31-1 354 355 802 GM2 (1-3 74 192 1,506 J6~~~~~~~~~~~6 LcOse3Cer (1-4 6190 5964 12,900 ml~~ ~ ~ ~ ~ ~~v nLcOse4Cer cal-3 876 1086 470 The complete incubation mixtures contained the same components as described for the glycolipid glycosyltransferase assays except that the indicated substrates were used. The rates of the reactions were proportional to the protein concentrations in the given ranges and FIG. 1. Phase contrast micrographs of TSD SV4O-transformed remained constant with time of incubation up to 2 hr. Breakdown of glial cells (a) and the same cells passaged through soft agar (b). The UDP-[14C]galactose was less than 7% and was tested with the TSD cells were maintained on Eagle's minimal essential medium (F-iS; homogenate under our reaction conditions by paper chromatography Difco) as described in the text. (X200.) (32), in ethanol/1 M ammonium acetate (7:3, vol/vol) at pH 7.4. Downloaded by guest on October 2, 2021 4272 Biochemistry: Basu et al. Proc. Natl. Acad. Sci. USA 76 (1979)

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C- - a Ar.aa m 0 - 2.0 4.0 6.0 8.0 Protein, mg/ml Protein, mg/ml

FIG. 2. Effect of TSD cell homogenate on GalT-3 of ECBM FIG. 3. Effect of protein concentration on sialyltransferase ac- fraction P3. The incubation conditions were the same as described tivities. Conditions were the same as described in the text for gly- in the text for glycolipid glycosyltransferase assays except that the colipid:sialyltransferase assays, except that the indicated quantities concentration of ECBM was varied as indicated. 0, Activities with of TSD cell homogenate fractions were used. Incubation was at 370C the ECBM fraction; A, activities with the ECBM fraction in the for 2 hr, and the mixtures were assayed by paper chromatography as presence of 100 Mg of TSD cell homogenate protein; activities with described in the text. [14CSialic acid incorporation into the following ECBM fraction in the presence of 200 Mtg of TSD cell homogenate three substrates was tested: *, GgOse4Cer; A, nLcOse4Cer; and 0, protein. lactosylceramide (LacCer) [Gal(ll1-4)Glc-Cer].

6.4), 10; MgCl2, 0.125; and CMP-[14C]AcNeu. 0.027 (2.47 X 106 2). The effect of embryonic age on three different chicken brain cpm/mmol). glycolipid galactosyltransferases was reported previously (31). The standard reaction mixture (55 sil) for the fucosyltrans- The activity of UDP-galactose:2-hydroxyceramide (31-1)- ferase assay contained the following (in ,tmol): glycolipid, 0.05; galactosyltransferase showed a sharp rise corresponding to the detergent G-34-A (Atlas Chemical), 0.2 mg; cacodylate/HCI period of active myelination, followed by a sharp drop at em- buffer (pH 6.35), 10; MgCl2, 0.125; GDP-L-[14C]fucose, 0.015 bryonic age of 19-21 days. Recently Costantino-Ceccarini and (2.7 X 106 cpm/Amol); and bovine spleen Golgi-rich membrane Suzuki (33) showed the presence of an inhibitor of this enzyme fraction, 0.1-0.2 mg of protein. in adult rat brain. In order to obtain GaIT-S activity devoid of any inhibitor, we used 12- to 14-day-old embryonic chicken RESULTS brain for these studies. As shown in Fig. 2, even in the presence Absence of Inhibitors of Glycolipid Galactosyltransferase of 200 ,tg of TSD cell homogenate protein per 100-Ml incuba- Activities in TSD Cells. The galactosyltransferase activities tion volume, there was almost no inhibition of GM1a formation. were compared in a TSD transformed line (Fig. la) and a These results suggested that GalT-3 activity was absent or very highly tumorigenic TSD transformed clone obtained by passage low in TSD cells in comparison to GalT-4. There was also no through soft agar (Fig. lb). Of all the substrates tested (Table inhibitor of GalT-3 activity in these cell lines. 2), LcOse3Cer and nLcOse4Cer had the highest activities in Comparison of Glycolipid Sialyltransferase Activities in both clones. GaIT-3 activity, which catalyzes the synthesis of TSD Cells. The results of the transfer of to various GMla ganglioside from GM2, was significantly low in both cell potential glycolipid acceptors showed the biosynthesis in vitro lines. We also determined the GalT-3 activity (10) in a purified of GM3, GD1a, and GM1b and also of sialylneolactotetraosyl- embryonic chicken brain membrane ECBM preparation from [GM1b(GlcNAc)] in TSD cells (Table 3). With 12- to 14-day-old embryos (Table 2). In order to determine nLcOse4Cer as substrate the Vmax values varied between 0.3 whether TSD cells do contain an inhibitor of GalT-3, we com- and 0.5 (nmol/ml per 2 hr) at protein concentrations of 2.5 and pared the same enzyme activity of embryonic chicken brain 7.5 mg per ml, respectively (Fig. 3). Under similar conditions in the presence and the absence of the TSD homogenates (Fig. the Vmax values of GgOse4Cer remained constant at all protein

Table 3. CMP-AcNeu:glycolipid sialyltransferase activities in TSD transformed cerebrum cells Table 4. Inhibition of brain sialyltransferases by TSD transformed cerebrum cell homogenate [14C]AcNeu incorporated, [14C]AcNeu incorporated, Acceptor pmol/mg protein per 2 hr nmol/ml per 2 hr (0.5 mM) TSD-agar TSD ECBM (P3) Acceptor ECBM ECBM % Lactosylceramide 128 385 935 (0.5 mM) (P3) TSD + TSD inhibition GM3 ganglioside 158 77 697 LacCer 1.58 0.38 1.98 0 GMla ganglioside 201 361 1113 nLcOse4Cer 4.50 0.43 3.0 38 GgOse4Cer 635 892 4262 GgOse4Cer 11.10 1.31 8.0 36 nLcOse4Cer 283 303 1933 The incubation conditions were the same as described for Table The reaction mixtures contained the same components as described 3 except that the TSD cell homogenate (250 jig of protein per incu- in the text except that the indicated substrates were used. The ECBM bation volume) was used as the source of inhibitors. A 9-day-old was from 9-day-old embryos. The rates of the reactions remained ECBM preparation (440 ,gg of protein per incubation volume) was constant with time of incubation up to 2 hr and were proportional to used as the source of sialyltransferase activities. Under the incubation protein concentration in the given ranges. Under the incubation conditions (0.05% 2-mercaptoethanol), hydrolysis of CMP-[14C]sialic conditions [in the presence of 0.025-0.05% 2-mercaptoethanol (32)] acid was less than 5%. The rates of the reactions remained constant hydrolysis of CMP-[14C]sialic acid was less than 3% with the TSD cell for 2 hr and were proportional to the protein concentration of ECBM homogenate. and TSD enzyme fractions in the given ranges. Downloaded by guest on October 2, 2021 Biochemistry: Basu et al. Proc. Natl. Acad. Sci. USA 76 (1979) 4273 Table 5. Inhibition of bovine spleen fucosyltransferases by TSD LcOse3Cer (al-3)fucosyltransferase (34) in the TSD cell ho- transformed cell homogenate mogenate. Moreover, the reaction catalyzed by the BSGM [14C]Fuc incorporated, fraction was inhibited (100%) by the TSD homogenate (Table nmol/ml per 45 min 5). Acceptor BSGM % (1.4 mM) Linkage BSGM TSD + TSD inhibition DISCUSSION The results of these experiments show clearly that there is very LcOse3Cer al-3 3.5 0 0 100 little activity and no inhibition of GalT-3 in TSD cells. TSD cells nLcOse4Cer al-2 41.2 1.6 9.9 75 passaged through soft agar have an even lower activity of the nLcOse5Cer al-2 52.7 4.0 13.2 77 enzyme. On the other hand, the activity of GaIT-4 (35) is 30- The incubation mixtures contained the same components as de- to 80-fold higher than that of GalT-3. In a separate experiment, scribed in the text except that the indicated N-acetylglucosamine- the GalT-4 activity of ECBM fraction was inhibited less than containing glycolipid substrates were used. The rates of the reactions It can be remained constant with the time of incubation up to 45 min and were 5% in the presence of the TSD cell homogenate. proportional to the protein concentration (97 jig of BSGM protein speculated that TSD transformed cells have a genetic block in per incubation volume). TSD homogenate was 250 Mig per incubation the synthesis of GalT-3 and contain no inhibitors of GalT-3 or volume. Under the reaction conditions hydrolysis of GDP-[14C]fucose GalT-4. The immediate question remaining to be answered is was less than 5% (tested in the presence and absence of 5 mM EDTA). whether the nLcOse4Cer thus formed in TSD cells is utilized for blood group-active glycolipids (e.g., H-active) by FucT-2 or is converted to a GlcNAc-containing ganglioside, AcNeu- concentrations. These results raised the question whether the nLcOse4Cer, by (a2-3)sialyltransferase (SAT-3) (see Fig. 4). decreases in maximum velocities are due to the presence of a It has also been reported that the concentration of a GlcNAc- specific inhibitor of the sialyltransferase in TSD cells. The containing ganglioside is elevated in these TSD transformed membrane fraction isolated from 7- to 12-day-old embryonic cells (14). chicken brain had the highest CMP-AcNeu:nLcOse4Cer si- Although low but significant activity of SAT-3 is observed alyltransferase activity (unpublished). The membrane fraction in TSD and TSD cells passaged through soft agar, it is also ob- isolated from 9-day-old embryonic chicken brain was used for served that the Vmax with GgOse4Cer is 2 to 3 times higher than a mixing experiment (Table 4). It appeared that the biosynthesis with nLcOse4Cer (Table 3). A competition experiment (refs. in vitro of GM3 was not inhibited, whereas almost similar in- 15, 16; unpublished) suggests that these two reactions are hibition was observed with nLcOse4Cer and GgOse4Cer. probably catalyzed by a single protein present in the BSGM Inhibition of Glycolipid (al-2)Fucosyltransferase Activity fraction. Inhibition of this sialyltransferase activity (SAT-3) at in TSD Cells. The conversion of nLcOse4Cer to H1 glycolipid higher TSD protein concentrations (Fig. 3) suggests that an by the TSD cell homogenate was tested in vitro in the presence inhibitory protein may be present in this cell extract. The pos- and absence of the bovine spleen Golgi-rich membrane (BSGM) sibility of excess hydrolysis of CMP-AcNeu at higher protein fraction (18) and was compared with values obtained from the concentrations has been eliminated because of the presence of reaction catalyzed by the BSGM fraction alone (Table 5). The 0.1% 2-mercaptoethanol (32) in the incubation mixture. activities of (al-2)fucosyltransferase (FucT-2) with both Until now, skin fibroblasts from TSD patients have been used nLcOse4Cer and nLcOse5Cer were much lower than the ac- to study the disease (36), and it is important to note that those tivities observed in the BSGM fraction. However, 75-77% in- skin fibroblasts do not accumulate GM2 ganglioside (9). Our hibition of FucT-2 was observed when the TSD cell homoge- present report and the studies of brain ceramide hexosides by nate was added to incubation mixtures containing the BSGM Suzuki and Chen (37) suggest that specific biosynthetic and fraction. There was no detectable activity of GDP-Fuc: degradative steps of brain ganglioside may be de-

| al7-GIc-Cer CMP-AcNeu Gal-Gic-Cer UDP- GlcNAr, GcNAc-GI-Glc-Cer| AcNeu SAT-I CMP- AcNeu - Lactosylceramide UDP- Gal LcOs , 3Cer SAT-;/ UDP- GaINAN\GM3) GaIT-44 GalNAc-Val-Glc-Cer | Gal-GIcNAc-Gal-Glc-Cer | AcNeu CMP- AcNeu nLcOse Cer 4 (GD3) UDP-Gal (GM2) SAT-3 GDP-Fuc GQIT-3t ? Gal-GlcNAc- Gal-Gic-Cer FucT-2 AcNeu Gal-GaINAc-Gal-Glc-Cer _ AcNeu [GMlbGlcNAc)] CMP- AcNeu1 (GMIa) Yal-GicNAc-Gal-Glc- Cer SAT-4 Fuc

?|l- GoINAc- al-Gtc-Cer 1 HI AcNeu AcNeu j (GDIO) FIG. 4. Proposed pathways for ganglioside biosynthesis in TSD SV40-transformed cultured cells. Downloaded by guest on October 2, 2021 4274 Biochemistry: Basu et al. Proc. Natl. Acad. Sci. USA 76 (1979)

leted in transformed TSD cells. However, the subject is now 16. Chien, J.-L. (1975) Dissertation (Univ. Notre Dame, Notre Dame, open to direct experimental test with untransformed TSD cells. IN). A permanent untransformed TSD cerebrum cell line is not 17. Basu, S., Basu, M., Presper, K. A., Tang, A. & Hoffman, L. M. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 1767. available at present. 18. Basu, S., Basu, M. & Chien, J.-L. (1975) J. Biol. Chem. 250, 2956-2962. We thank Drs. Larry Schneck and Daniel Amsterdam for helpful 19. Risser, R. & Pollack, R. (1974) Virology 59,471-489. discussions. This work was supported by U.S. Public Health Service 20. Kean, E. L. & Roseman, S. (1966) J. Biol. Chem. 241, 5643- Grants CA-14764 and NS-09541 and a grant-in-aid from Miles Labo- 5650. ratories to S.B. 21. Schachter, H., Ishihara, H. & Heath, E. C. (1972) Methods En- zymol. 28, 285-287. 22. Basu, M. & Basu, S. (1973) J. Biol. Chem. 248, 1700-1706. 1. Klenk, E. (1939) Z. Physiol. Chem. 263, 128-143. 23. Moskal, J. R., Chien, J.-L., Basu, M. & Basu, S. (1975) Fed. Proc. 2. Svennerholm, L. (1962) Biochem. Biophys. Res. Commun. 9, Fed. Am. Soc. Exp. Biol. 34,645. 436-441. 24. Moskal., J. R. (1977) Dissertation (Univ. Notre Dame, Notre 3. Suzuki, K., Suzuki, K. & Samoshito, S. (1969) j. Neuropathol. Exp. Dame, IN). Neurol. 28, 25-73. 25. Li, Y. T. & Li, S. C. (1972) Methods Enzyniol. 28,714-720. 4. Svennerholm, L. (1966) Acta Pediatr. Scand. 55,546-562. 26. Basu, S., Basu, M., Moskal, J. R., Chien, J.-L. & Gardner, D. A. 5. Schneck, L. & Volk, B. W. (1967) in Proceedings of the Third (1976) in Glycolipid Methodology, ed. Witting, L. A. (American International Symposium on the Cerebral , Oil Chemist's Society, Champaign, iL), pp. 123-139. eds. Aronson, S. M. & Volk, B. W. (Pergamon, New York), pp. 27. Svennerholm, L. (1969) in Comprehensive Biochemistry, eds. 403-411. Florkin, M. & Stotz, E. H. (Elsevier, Amsterdam), Vol. 18, pp. 6. Schneck, L., Adachi, M. J. & Volk, B. W. (1972) Pediatrics 49, 201-227. 342-353. 28. Bjorndal, H., Hellerqvist, C. G., Linderberg, B. & Svensson, S. 7. Wyatt, P. R., Cox, D. M. & Hoogstraten, J. (1978) Pediatr. Res. (1970) Angew. Chem. Int. Ed. Engl. 9,610-619. 12,310-313. 29. Basu, S., Kaufman, B. & Roseman, S. (1973) J. Biol. Chem. 248, 8. Cotlier, E. (1972) Clin. Chim. Acta 38,233-234. 1388-1394. 9. Dawson, G., Matalon, R. & Dorfman, A. (1972) J. Biol. Chem. 30. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. 247,5951-5958. (1951) J. Biol. Chem. 193, 265-275. 10. Okada, S. & O'Brien, J. S. (1969) Science 165, 698-700. 31. Basu, S., Schultz, A. M., Basu, M. & Roseman, S. (1971) J. Biol. 11. Basu, S., Kaufman, B. W. & Roseman, S. (1965) J. Biol. Chem. Chem. 246, 4272-4279. 240,4115-4117. 32. Kean, E. L. (1972) Methods Enzymol. 28,983-990. 12. Hoffman, L. M., Amsterdam, D. & Schneck, L. (1976) Brain Res. 33. Costantino-Ceccarini, E. & Suzuki, K. (1978) J. Biol. Chem. 253, 111, 109-117. 340-342. 13. Schneck, L., Hoffman, L. M., Amsterdam, D., Brooks, S. E. & 34. Presper, K. A., Basu, M. & Basu, S. (1978) Proc. Natl. Acad. Sci. Pinkett, B. (1976) in Current Trends in Sphingolipidoses and USA 75, 289-293. Allied Disorders, eds. Volk, B. W. & Schneck, L. (Plenum, New 35. Basu, M. & Basu, S. (1972) J. Biol. Chem. 247, 1489-1495. York), pp. 495-507. 36. Kolodny, E. H., Milunsky, A. & Sheng, G. S. (1973) Birth Defects 14. Hoffman, L. M., Brooks, S. E., Amsterdam, D. & Schneck, L. Orig. Artic. Ser. 9, 130-135. (1978) Trans. Am. Soc. Neurochem. 9,206 (abstr.). 37. Suzuki, K. & Chen, G. C. (1967) J. Lipid Res. 8, 105-113. 15. Chien, J.-L., Basu, M. & Basu, S. (1974) Fed. Proc. Fed. Am. Soc. 38. IUPAC-IUB Commission on Biochemical Nomenclature (1977) Exp. Biol. 34, 1225. 12, 455-468. Downloaded by guest on October 2, 2021