THE JOURNAL OF BIOLOGICAL CKEMI~TRY Vol. 248, No. 4, Issue of February 25, pp. 1388-1394, 1973

Printed in U.S.A.

Enzymatic Synthesis of Glucocerebroside by

a Glucosyltransferase from Embryonic Chicken Brain*

(Received for publication, July 13, 1972)

SUBHASH BASU,~ BERNARD KAUFMAN,~ AND SAUL ROSEMAN

From the McCollum-Pratt Institute and the Department of Biology, The Johns Hopkins Unitjersity, Baltimore, Maryland 61.218

SUMMARY

A glucosyltransferase that catalyzes the synthesis of glu- cosylceramide (glucocerebroside) from ceramide (N-acyl- sphingosine) and UDP-glucose was isolated from 13- to 14-day-old embryonic chicken brain. The reaction is the following:

Ceramide + UDP-glucose -+ glucosylceramide (+ UDP)

Ceramide was the most effective lipid acceptor, and a variety of potential donors could not replace UDP-glucose. The K, values for ceramide and UDP-[%Jglucose were 0.08 mu and 0.12 XIIM, respectively. Metal ions did not stimulate the reaction, and it was not inhibited by ethylenediaminetetra- acetate. The particulate preparation of UDP-glucose: cer- amide glucosyltransferase showed an absolute requirement for detergent, and optimal activity was obtained with a mix- ture of Cutscum and Triton X-100.

The glucosyltransferase was detectable in brains from chick embryos as early as 7 days old; maximal activity was detected between 13 and 17 days of embryonic development. The enzyme catalyzes the first step in the synthesis of gan- gliosides.

I-O-b-n-Glucopyranosylceramide (glucocerebroside) is widely distributed in normal and pathological tissue, including normal human serum (l), plasma (2, 3), erythrocytes (4), kidney (5, 6), and aortic tissue (7), as well as in the central nervous system (8- 10). It is the major cerebroside found in the spleen of patients with Gaucher’s disease (11-13). Since glucose is the first sugar directly attached to ceramidel in oligoglycosylceramides and

* These studies were supported by Grant AM-9851 from the National Institutes of Health and Grant P-544 from the American Cancer Society. Contribution No. 712 from the McCollum- Pratt Institute.

1: This work was carried out under t,he tenure of a research fellowship from the Helen Hay Whitney Foundation. Present address. Denartment of Chemistrv. Biochemistrv and Bionhvsics Program, University of Notre Dame, Notre Dame, Indiana 4g556.

8 Present address, Department of Biochemistry, Duke Uni- versity, Durham, North Carolina 27706.

1 The nomenclature used is: ceramide, N-acylsphingosine, where the acyl group contained the following fatty acids: Cls,o,

gangliosides, glucocerebroside is thought to be the precursor of the more complex glycolipids (14).

Martensson et al. (15) have shown the incorporation of [r4C]- glucose into glucosyl- and lactosylceramide of intact human leukocytes, and more recently, Nishimura et al. (16) and Maker and Hauser (17) have shown incorporation of [r4C]glucose into different gangliosides by adult guinea pig and young rat brain slices.

We previously reported the stepwise in z&o synthesis of gan- gliosides from lactosylceramide with a membrane-bound enzyme system isolated from embryonic chicken brain. Each of the steps in the pathway required the transfer of a monosaccharide unit from its respective sugar nucleotide to the terminal sugar of a specific oligoglycosylceramide (18-24). Each reaction was catalyzed by a specific glycosyltransferase present in the em- bryonic brain enzyme system. This paper describes the proper- ties of a glucosyltransferase from embryonic chicken brain that catalyzes the synthesis of glucocerebroside from ceramide and UDP-glucose. Preliminary accounts of these findings have been presented (25, 26) ; for purposes of clarity and convenience to the reader, some of the previously published results have been in- cluded in the present paper.

EXPERIMENTAL PROCEDURE

Materials

The following materials were obtained from commercial sources: Bicine,2 Tricine, and HEPES (Calbiochem) ; Whatman SG-81 silica gel-impregnated paper (W. and R. Balson, Ltd., Maidstone, Kent) ; Unisil, silicic acid (Clarkson Chemical Com- pany, Williamsport, Pennsylvania) ; nn-sphingosine and DL- dihydrosphingosine (Miles Laboratories, Inc., Elkhart, Indiana); hexokinase and glucose-6-P dehydrogenase (C. F. Boehringer and Sons, Mannheim, Germany). The following compounds were all uniformly labeled in the sugar moiety : UDP-[i%]glucose, UDP-[i4C]galactose, and [i4C]glucose (New England Nuclear) ;

43%, c16:1, 4.2%; CH:O, 19.3%; CIS:I, 5.1%; CU:O and G:I, 22.0%; glucocerebroside or gluoosylceramide, I-O-p-n-glucopyranosyl- ceramide. Unless otherwise specified, all sugars were of the D

configuration and glycosides were pyranesides. 2 The abbreviations used are: Bicine, (HOCHzCH&N

CH&OO-; Tricine (HOCHz),CNHCH&OO-; HEPES, +/-\

HOCHzCHzN NCHzCHzSOs(27). H\-/

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Analytical Procedures--Protein was determined by a modifica- tion of the method of Lowry et al. (29) ; the purified radioactive product of the enzymatic reaction was methanolyzed by the pro- cedure of Kishimoto and Radin (30) ; sphingosine was determined by the spectrophotometric method of Lauter and Trams (31) ; [r4C]glucose by the coupled enzymatic reaction (32) with the use of hexokinase and glucose-6-P dehydrogenase; and fatty acids by gas-liquid chromatography (33).

Thin Layer Chromatography-Thin layer chromatography was used to identify and purify sphingosines, ceramides, and cera- mide mono- and oligosaccharides. The following solvents were used: Solvent I, chloroform-methanol-acetic acid (95: 5: 1) ; Solvent II, chloroform-methanol-water (65 : 25 :4) ; Solvent III, chloroform-methanol-water (60 : 17 : 2). The spots were visu- alized by exposure to iodine vapor or by spraying with O.O4ol, bromthymol blue reagent (in 0.01 N NaOH). The thin layer chromatographic plates were coated with Silica Gel G and ac- tivated for 30 to 45 min at 110-115” before use.

FIG. 1. Density gradient separation of embryonic chicken brain crude mitochondrial fraction. Fraction PI. mselin fraction; Fractions P1 and Pa, membrane fractions containing synapto- somes; Fraction P,, mitochondria and cell membranes. Figures at right are sucrose concentrations. Centrifugation was con- ducted at 105,000 x g for 2 hours in a Spinco swinging bucket SW 41 rotor at 4”.

Preparation of Enzyme-The membrane-bound glucosyltrans- ferase was prepared from 13- to 14-day-old embryonic chicken brains as described previously (26). All steps in the preparation of enzymes were conducted between 0’ and 4”. The crude mito- chondrial fraction was resuspended in sucrose solution and was applied to the top of a discontinuous sucrose gradient (Fig. 1). Four membrane fractions were observed, and glucosyltransferase activity was distributed in those fractions as follows: at the junction between 0.32 M and 0.75 M sucrose, Fraction PI (2%) ; between 0.75 M and 1.0 M, Fraction P1 (10%); between 1.0 M and 1.2 M, Fraction PI (82%); and the precipitate at the bottom of 1.2 M layer, rich in mitochondria, Fraction P4 (4%). Fraction Pa was used as the enzyme source unless otherwise specified; the specific activity of Pa was about 6-fold higher than that of the crude homogenate.

Nt, and applied to Whatman SG-81 silica gel paper impregnated with 1 $‘J sodium tetraborate (34). Ascending chromatograms were developed with Solvent III. The paper strips were dried, cut into l-inch segments, and counted in a toluene system in the Packard liquid scintillation spectrometer, model 3320 (RF values for glycosphingolipids were as follows: glucosylceramide, 0.64, galactosylceramide, 0.28; lactosylceramide, 0.08; ceramide, 0.94; sphingosine, 0.36; glucosylsphingosine, 0.12; galactosylsphingo- sine, 0.03). An alternative procedure was also used for separat- ing “C-labeled UDP-glucose and its degradation products, glu- cose and glucose-l-P, from the labeled lipid product. This method, which involved high voltage electrophoresis in 1.0% sodium tetraborate (21) on Whatman No. 3MM paper, was used when the labeled lipid product partitioned into the aqueous phase of the Folch wash, as in the case of glucosylsphingosine.

RESULTS

Enzyme Assay-Complete incubation mixtures contained the following components in final volumes of 0.1 ml: ceramide, 0.2 pmole; Cutscum-Triton X-100 (2: l)a, 0.6 mg; Bicine buffer, pH 8.1, 15 pmoles; UDP-[i4C]glucose, 0.1 pmole (3.0 to 5.0 X 10’ cpm per pmole); and enzyme fraction, 0.4 to 0.8 mg of protein. The mixtures were incubated for 1 hour at 30”, and the reactions were stopped by adding 2.5 pmoles of EDTA (pH 7.0) and 5 moles of KC1 in 20 ~1 of water, followed by 0.5 ml of chloroform- methanol (2:l). After mixing in the Vortex mixer, the lower layer (0.35 ml) was washed once with 0.2 ml of theoretical upper phase (chloroform-methanol-O.1 M KCl, 3 :47 :48), dried under

a Lipid substrates and detergents were first dissolved in chloro- form-methanol (2:1), transferred to the incubation tubes, and dried in a vacuum at room temperature.

Requirements for Enzyme Activity-The incorporation of [14C]- glucose into ceramide did not require Mg++ or Mn++ (Table I), and EDTA did not inhibit the reaction. Omission of detergent, however, resulted in a marked decrease in the rate. Neutral detergents such as Tween 20, Tween 80, and Triton CF-54 did not substitute for Cutscum or Triton X-100, and a mixture of the latter gave better results than either alone. An optimal concentration of 6 to 12 mg of detergent mixture (Cutscum to Triton X-100, 2: 1) per ml was determined as shown in Fig. 2. Under these conditions, product formation was proportional to protein concentration in the range 4.0 t,o 9.0 mg per ml as shown in Fig. 3, and to time of incubation up to 90 min (Fig. 4) at 30”. The enzyme was completely inactivated by incubating for 15 min at 37” in the absence of UDP-glucose (2.2% of optimal ac-

ADP-[r4C]glucose, CDP-[l%]glucose, and GDP-[l*C]glucose (International Chemical and Nuclear Corporation, Irvine, Cali- fornia).

Ceramide and n-sphingosine were isolated from bovine lungs by the method of Tipton (28). We are grateful to Dr. Norman S. Radin for his gift samples of psychosines and Gaucher spleen glucosylceramide.

Detergents--Triton X-100 (polyethoxy alkyl phenol ether) was purchased from Packard Instrument Company, and Triton CF- 54 (alkyl phenoxy polyethoxy ethanol) from Rohm and Haas Company, Philadelphia. Tween 20 (polyoxyethylene 20-sorbi- tan monolaurate) and Tween 80 (polysorbate) were obtained from Atlas Chemicals Division of ICI America. Cutscum (iso- octyl phenoxy polyoxy ethanol) was a product of Fisher.

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TABLE I

Requirements fol, UDP-giucose:ceramide glucosyltransferase Complete incubation mixtures contained the following com-

ponents in final volumes of 0.1 ml: ceramide, 0.1 pmole; Cutscum, 0.4 mg; Triton X-100, 0.2 mg; glycylglycine buffer, pH 8.1, 20 pmoles; UDP-[Wlglucose (5.3 X lo6 cpm per pmole), 0.11 rmole; enzyme, Fraction PI, 0.60 mg of protein. Mixtures were incu- bated for 60 min at 30” and were assayed by the chromatographic method described in the text.

Incubation mixtures [‘“ClGlucocerebroside

c@z/mg $rolein/hr

Complete................................. Minus ceramide......................... Minus detergents. . Plus MgClt (0.5 rmole) Plus MnClz (0.5 pmole). Plus EDTA (2.5 pmoles) Minus active, plus heat-inactivated

enzyme (3 min at 100”).

6042 1407

186 6445 6040 5631

75

0 1.2 w +

n

g 0. a

0; f ‘0

- 0.6 wx

:: E v 20.4 3- J

0.0 2.4 4.8 7.2 9.6 12.0 14.4 18.0

CUTSCUM :TR ITON x-100 ( mg/ ml ) (2: I )

FIG. 2. Effect of detergent concentration on the rate of reac- tion. Incubation mixtures contained the following components in final volumes of 0.1 ml: ceramide, 0.1 pmole; glycylglycine buffer, pH 8.1, 20 rmoles; UDP-[Wlglucose (3.4 X IO6 cpm per rmole), 0.1 rmole; enzyme, Fraction Pt, 0.6 mg of protein; and Cutscum and Triton X-100 (2:1, w/w) as indicated. Mixtures were incubated at 30” for 45 min and were assayed by the chroma- tographic method described in the text. The values given in the figure were corrected for endogenous values, 257 to 741 cpm per O.l-ml incubation. In this and all subsequent figures, the values in the ordinates represent 1% product per O.l-ml incubation mixture.

tivity) . The rate of incorporation of glucose into endogenous

and exogenous (ceramide) substrates was found to be markedly depressed at temperatures above 32”, as shown in Fig. 5. A pH optimum of 7.9 (measured after incubation) was exhibited in i%cine buffer (Fig] 6).

The endogenous values, i.e. the quantities of labeled product formed in the absence of added ceramide, were substantial and are given in each experiment. In evaluating these results, it is important to note that the enzymatic assay involved chromato- graphic separation of the labeled product from other known glycolipids, including galactosylceramide. The product formed from the endogenous acceptor showed behavior identical with

0 e -

0.0 I .r3 3.6 5.4 z2

PROTEIN ( mg /ml)

FIG. 3. Effect of protein concentration on the rate of reaction. Conditions were the same as described in Fig. 2 except that the concentration of protein was varied as indicated and 0.6 mg of detergent was used per 0.1 ml of incubation; mixtures were as- sayed by the chromatographic method described in the text. The values given in the figure were corrected for endogenous values, 92 to 555 cpm per O.l-ml incubation.

0 d -

0 I5 30 45 60 75 90

TIME ( MINUTES )

FIG. 4. Effect. of incubation time on the rate of reaction. Con- ditions were the same as those described in Fig. 3 except that 0.6 mg of protein was used per O.l-ml incubation, and the tubes were incubated at 30” for the indicated times; mixtures were assayed by the chromatographic method described in the text. The values given in the figure were corrected for endogenous values, 64 to 840 cpm per O.l-ml incubation.

authentic glucosylceramide when subjected to chromatography on SG-81 paper, and Silica Gel G thin layer plates. In addition, substantial quantities of ceramide were detected in the enzyme preparation, Pa. These results therefore indicate that the in- corporation of glucose into endogenous acceptor yielded gluco- sylceramide, and the enzymatic activity being measured was, in fact, the UDP-glucose:ceramide glucosyltransferase.

Sub&ate Speci$ci@-The most active substrate was ceramide (Table II), The rate of incorporation of [14C]glucose from UDP- [Wlglucose into sphingosine or dihydrosphingosine was only 23 to 30% of that obtained with ceramide, and the products from the sphingosines appeared to be the corresponding psychosines

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- CERAMIDE 0-0 ENDOGENOUS

0

INCUBATION TEMPERATURE PC)

FIG. 5. Effect of temperature on the glucosyltransferase reac tion. Conditions were the same as those described in Fig. 2 except that the incubation mixtures were maintained for 30 min at the indicated temperatures in the presence and absence of added ceramide.

f g.“l a

E 7. 5 -

k 0 6.0- v- Z’:

- 0 4.5- WX

:: E 0 ,p3.0- 3-

6.0 8.0 9.0

PH FIG. 6. Effect of pH on the rate of formation of glucosylcer-

amide. Incubation mixtures were the same as those described in Table II (with ceramide as acceptor) except that 0.15 pmole of the following buffers were used: A, Bicine-sodium hydroxide; q , HEPES-sodium hydroxide; A, Tricine-sodium hydroxide; 8, glycylglycine-sodium hydroxide; 0, Tris-HCI; A, potassium phosphate; 0, cacodylate-HCl; and l , sodium acetate-acetic acid. The mixtures were incubated at 30” for 60 min and were assayed by the chromatographic method described in the text. The values given in the figure were corrected for endogenous values, 242 to 3308 cpm per O.l-ml incubation.

(glucosylsphingosines). The effects of varying the ceramide and

sphingosine concentration on the rate of reaction are shown in

Figs. 7 and 8, respectively. The K, values for ceramide and

sphingosine were 0.08 mM and 5.0 mM, respectively. Donor Specijicity-None of the following compounds could

substitute for UDP-[14C]glucose: [r4C]glucose, [14C]glucose-l-P, ADP-[r4C] glucose, GDP-[14C] glucose, or UDP2[14C] galactose (Table III). CDP-[14C]glucose, and UTP plus [r4C]glucose-l-P,

TABLE II

Substrate specificity of embryonic chicken brain, glucosyltransferase

Incubation mixtures contained the following components in final volumes of 0.1 ml: Bicine buffer, pH 8.1, 15.0 pmoles; Cuts- cum, 0.4 mg; Triton X-100, 0.2 mg; UDP-[Wlglucose (specific activity, 5.3 X lo6 cpm per pmole), 0.11 rmole; enzyme, Fraction Pt, 0.6 mg of protein; and 0.2 pmole of each of the indicated acceptors. The mixtures were incubated at 30” for 60 min and were assayed by the chromatographic method (glucosylceramide) and high voltage paper electrophoresis (glucosylsphingosine) as described in the text.

‘4C Product

Acceptors’ Glucosyl- ceramide

Glucosyl- sphingosine

None.................................. 4,310 391 Ceramide.............................. 13,100 505 nn-Sphingosine 3,220 2,990 nn-Dihydrosphingosine. 2,850 3,930 Ceramide $ nL-sphingosine. 9,310 3,230 Ceramide + nn-dihydrosphingosine. 11,900 1,850

a The following compounds showed no activity as acceptors: glucosylceramide, galactosylceramide, lactosylceramide, N- acetylneuraminyllactosylceramide (Gn3), N-acetylgalactosami- nosyllactosylceramide, and Tay-Sachs ganglioside (Gm).

n 7.2 1

0.0 0.4 0.0 I .2 I .6 2.0 2.4 2.8

S, CERAMIDE (m_M)

36

30

‘5

1

24 7 0 -

18 x

<

12 z

6

0

FIG. 7. Effect of ceramide concentration on the rate of forma- tion of glucocerebroside. Incubation mixtures were the same as in Table II, except that varying concentrations of ceramide were used. Incubations were conducted for 65 min at 30” and the chromatographic method described in the text was used for assay. The values given in the figure were corrected for endogenous value, 2591 cpm per O.l-ml incubation.

gave 5 to 7% of the optimal activity. The effect of varying the UDP-[i4C]glucose concentration on the rate of reaction is shown

in Fig. 9 and the calculated K, value was 0.12 mM. Effect of Embryonic Age-The specific activity of the enzyme

varied with the age of the embryo as shown in Fig. 10. Unlike

the galactosyltransferase that catalyzed the biosynthesis of galactosylceramide, which was detected late in embryonic de- velopment (after the embryo was 15 days old), the glucosyl- transferase active with ceramide exhibited significant activity even when the embryo was 7 days old. The optimal activity

was observed in 13-day-old embryonic chicken brain. Tissue Survey-A survey of various animal brains for the

galactosyltransferase is presented in Table IV. The glucosyl-

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2.80

[s] , Sphingosine (mM) FIG. 8. Effect of nksphingosine concentration on the rate of formation of glucosylsphingosine. Incubation mixtures were the same

as in Table II except that varying concentrations of nL-sphingosine were used. Incubations were conducted for 65 min at 30” and the chromatographic method was used for assay. The values given were corrected for endogenous value, 277 cpm per O.l-ml incubation.

TABLE III Donor specijicity of UDP-g2ucose:ceramide glucosyltransferase

Conditions were the same as those described in Table I except that the nucleotide sugars were varied. The mixtures were incubated at 30” for 60 min, and were assayed by the chromato- graphic method as described in the text.

Specific activity of donor DOIUX concentration

“C-Sugar incorporateda

cpmiwde /mole

UDP-[Wlglucose, 5.3 X 106. 0.11 6041 UDP-[“Clgalactose, 3.6 X 106. 0.09 107 ADP-[Wlglucose, 2.2 X 106. 0.10 115 CDP-[‘Slglucose, 2.6 X 106. 0.10 406 GDP-[%]glucose, 3.1 X 106. 0.10 188 [l%]Glucose, 0.58 X 106. 0.20 120 [%]Glucose-l-P, 1.3 X 106.. 0.10 135 [l%]Glucose-1-P,b 1.3 X 106. 0.10 301

a The endogenous value with UDP-[14C]glucose was 1407 cpm, and with the other radioactive donors varied from 100 to 300 cpm.

b Plus 0.08 pmole of UTP.

transferase activities with ceramide were high in the young rat,

guinea pig, and fetal pig brains. The activities observed in the

adult brains were very low, but are considered significant in view

of the specificity of t’he assay. Isolation, Characterization, and Analysis of 14C Product-The

radioactive glucocerebroside was isolated from a large scale incu- bation mixture which contained the following components in a final volume of 16 ml: ceramide, 28 mg; mixture of Cutscum and Triton X-100 (2:1), 120 mg; Bicine buffer, pH 8.1, 2.0 mmoles; UDP-[14C]glucose (3.4 x lo6 cpm per pmole), 20 pmoles; enzyme, membrane Fraction Pa of the 14-day-old chicken embryos, 8.5 ml (113 mg of protein). After 4 hours at 30°, the reaction was stopped by the addition of 100 ml of chloroform-methanol (2: I), 2 ml of 0.5 M KCl, and 0.5 ml of 0.25 M EDTA. The lower chloroform layer was washed twice with theoretical upper phase containing chloroform-methanol-O.1 M KC1 (3:47:48). The 14C product (1.0 rmole) in the lower phase was applied equally over six sheets of Silica Gel SG-81 paper, which were developed

P 9.0

F a cc 7.5 -

izy

g ,6.0- a

z t.7 T

-’ 45-. wo‘

%

mx /

-15;

>

E? g3.o-o Z”

/ :

IO’

I 3

W

3 ‘.5y Km q 1.2 x 10m4M 5

” i *’ I I I I I I ’ 0

0.4 0.8 1.2 I .6 2.0 2.4 2.6

S, UDP-Glu (mM_l

FIG. 9. Effect of UDP-[Wlglucose concentration on the rate of formation of [‘%]glucosylceramide. Incubation mixtures were the same as in Table II (with ceramide as acceptor) except that the indicated concentrations of UDP-[‘%]glucose were used. The mixtures were incubated for 65 min at 30” and were assayed by the chromatographic method described in the text. The values given in the figure were corrected for endogenous values, 376 to 3600 cpm per O.l-ml incubation.

in the ascending manner with Solvent III. The radioactive areas of the chromatograms were eluted with Solvent II, dried, and a total of 0.66 pmole of 14C product was resuspended in chloroform-methanol (98 : 2)) and applied to a Unisil column (1

x 20 cm, activated Unisil packed by gravity filtration in chloro- form). Increasing concentrations of methanol in chloroform were passed batchwise through the column (chloroform-methanol, 98:2, 80 ml; 92:8, 70 ml; 80:20,85 ml; 60 :40,90 ml) ; the [‘“Cl- glucocerebroside was found in the chloroform-methanol (92 :8) eluate, which was concentrated to dryness (recovery, 91%). The [14C]glucocerebroside was further purified by thin layer chromatography in Solvent II (yield 82 %). Chromatography in the three solvent systems described under “Methods” showed it to be homogeneous and the only 14C component detectable after hydrolysis was [Wlglucose. The [14C]glucose was identi- fied by paper electrophoresis and paper chromatography (35) and further characterized by the hexokinase, glucose-6-P de-

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0 9.6 -

:

; g 8.0 -

kis 0 a 6.4-

‘\

0 CD 2 E -

n’ 4.8- 0 z ‘0

sx z, ; 3.2 - o

&-

(1, 1.6 - / \

* - 0 .A 1 I

O.O ‘7 9 I I I I.. .

13 15 17 26AIYJLT

AGE (DAYS)

FIG. 10. Effect of embryonic age on UDP-glucose:ceramide glucosyltransferase activity. Incubation mixtures contained the following components in final volumes of 0.1 ml: ceramide, 0.1 pmole; Cutscum, 0.4 mg; Triton X-100, 0.2 mg; Bicine buffer, pH 8.1, 10 Hmoles; UDP-[Wlglucose (3.8 X lo6 cpm per pmole), 0.1 pmole; enzyme, Fraction Pa of embryonic chicken brain of differ- ent ages, 0.3 to 0.8 mg of protein. Mixtures were incubated for 60 min at 30”, and assayed by the chromatographic method described in the text. Under these conditions, the rate of the reaction re- mained constant with time of incubation and was proportional to protein concentration. The values given in the figure were cor- rected for endogenous values, 270 to 650 cpm per O.l-ml incubation.

TABLC IV

UDP-glucose:cerawGde glucosyltransferase activity in brain homogenates of different species

Conditions were the same as those described in Fig. 10 except that the enzyme preparations used were unfractionated brain ho- mogenates (1 g of t,issue per 6 ml of 0.32 M sucrose containing 0.001 M EDTA and 0.014 M mercaptoethanol) of different species. Mixtures were incubated for 90 min at 30” and were assayed by the chromatographic met.hod described in the text.

SOUICe Age [“ClGlucocerebroside

cpm/mg protcin/hr

Chicken 20-day-old embryo 1827 (667)~ 7 months 504 (188)

Rat 8 days 3034 (807) 1 month 1235 (416)

Pig 29.cm fetus 1279 (393) Adult 406 (232)

Guinea pig 3 days 568 (192) 1 month 524 (199)

Sheep Adult. 442 (235) Human gray matter 1 year 439 (321)

a Numbers in parentheses are the endogenous values obtained from an identical experiment without added ceramide as acceptor.

hydrogenase reaction. The radioactive glucocerebroside was analyzed directly for 14C and for its constituents after methanolysis followed by acid hydrolysis (30). The following molar ratios were obtained by the analytical methods described above: sphingosine, 1.00; glucose, 1.06; [14C]glucose, 0.83; total fatty acids, 1.30. The labeled product contained the following fatty acids: C16:0, 31.8%; C1gEa, 40.9%; C~Z:O, 14.7%; CB:O, 7.5%; C24:I, 5.1%. The fatty acid composition of the product there- fore differed from that of the exogenous substrate. This differ-

ence can be explained by the fact that about 20y0 of the isolated product is derived from endogenous ceramide or cerebroside (or both). This is shown by the ratio of glucose to 14C in the product as well as the rate and extent of incorporation of [**C]glucose into cerebroside in the presence and absence of exogenous acceptor. As shown in Table I and Figs. 2 to 4, 6,7,9, and 10, the incorpo- ration into endogenous acceptor may be as high as 30% of that obtained in the presence of the exogenous acceptor.

DISCUSSION

We have previously reported that complex gangliosides, such as disialoganglioside, are enzymatically synthesized by the step- wise addition of monosaccharides to the terminal sugars of the oligosaccharide chain of these glycolipids (18-24, 36). Since the first sugar in the oligosaccharide chains of the most commonly found gangliosides (37) and blood group-related glycosphingo- lipids (25) is glucose, a key step in the synthesis of these com- pounds is the one which yields glucosylceramide (glucocerebro- side). The present studies describe a glucosyltransferase which catalyzes the synthesis of this important compound.

The glucosyltransferase was found in a particulate fraction isolated from embryonic chicken brain. The fraction, Pa, which contains both cell membranes and is enriched in nerve-ending particles or synaptosomes (36), was previously shown to contain the various glycosyltransferases involved in the synthesis of gangliosides (22-26). Unlike the galactosyltransferase which catalyzed the synthesis of galactosylceramide (galactocerebro- side), and which appears late in embryonic development (38, 39)) the glucosyltransferase was detected in the youngest brain sample tested, from 7-day-old embryos, although increased activity was noted with embryonic age (Fig. 10). Thus the pattern of ac- tivity with respect to embryonic age was very similar to those observed with the other glycosyltransferases involved in ganglio- side synthesis (22-26).

Of a number of potential lipids tested for acceptor act,ivity, only sphingosine and dihydrosphingosine could part’ially replace ceramide. The activity of the sphingosines suggested the pos- sibility that the pathway of synthesis of glucocerebrosidd might be sphingosine + glucosylsphingosine --t glucocerebroside. Such a pathway has, in fact, been suggested for the synthesis of ga- lactocerebroside (40-42). However, work in this laboratory (26, 39) and other laboratories (43-48) suggests that galacto- cerebroside is synthesized by the following sequence: sphingosine + ceramide --) galactocerebroside. The ability of sphingosine and ceramide to act as acceptors for glucose may represent the action of one nonspecific or of two specific enzymes, a point which remains to be resolved. Nevertheless, the in vitro studies re- ported here provide evidence that the pathway of synthesis of glucoeerebroside is by glucosylation of ceramide; it is of course recognized that in vitro results may not reflect the in vivo path- way. Our evidence is as follows: (a) the enzyme preparation contains an endogenous acceptor which is quite active; and all of the available evidence suggests that the endogenous acceptor is ceramide,d rather than sphingosine (51) or dolichol monophos- phate (52). For example, the 14C product formed from the endogenous acceptor is stable on treatment with 0.1 K HCl at room temperature for 30 min. According to Behrens et al. (52) dolichol monophosphate glucose is completely hydrolyzed under

4 Recently, Bretthauer et al. (49) have reported the presence of endogenous ceramide in the UDP-glucose:ceramide glucosyltrans- ferase of yeast (Hanselzula holstii). Glucosylceramide containing Cla:o fatty acid in the ceramide moiety has been previously iso- lated by Kaufman et al. (50) from yeast (Hansenula ciferri).

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these conditions. The i4C product obtained from the endogenous acceptor shows the chromatographic properties of glucosylcera- mide and- not of glucosylsphingosine (psychosine). (b) Kinetic studies show that exogenously added ceramide is’ a much more effective acceptor than erylhro-m-sphingosine or dihydrosphingo- sine. The Vmax for ceramide is 5-fold greater (at 2.0 mM con- centration) and the K, is about 62-fold lower than the corre- sponding values for sphingosine. Despite our conclusion which delineates a pathway for glucosylceramide synthesis, the activity of the enzyme with the sphingosines is of interest and remains to be resolved. If the activity is that of a specific enzyme, the metabolic significance of the product, glucosylsphingosine (53) is unknown, and its potential function would require clarification.

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Subhash Basu, Bernard Kaufman and Saul RosemanEmbryonic Chicken Brain

Enzymatic Synthesis of Glucocerebroside by a Glucosyltransferase from

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