THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 2, Issue of January 25, PP. 342-350, 1970

Printed in U.S.A.

Specificity in Ceramide Biosynthesis from Long Chain Bases and Various Fatty Acyl Coenzyme A’s by Brain Microsomes*

(Received for publication, August 20, 1969)

PIERRE MORELL$ AND NORMAN S. RADIN

From the Mental Health Research Institute, University of Michigan, Ann Arbor, Michigan 48104

SUMMARY

The formation of ceramide from long chain bases and W-acyl coenzyme A’s by a mouse brain microsomal prepara- tion was investigated. Activity of the acyl-CoA:long chain base N-acyltransferase was measured as a function of time, acyl-CoA concentration, amount of amine, and microsomal protein concentration. The rate and extent of conversion of stearoyl-, lignoceroyl-, palmitoyl-, and oleoyl-CoA to ceram- ide were in the ratio of about 60:12:3:1, respectively. Since this ratio strongly resembles the relative distribution of these fatty acids in brain sphingolipids, we suggest that the transferase plays an important role in controlling the observed distribution. There was some specscity with respect to the base, particularly with lignoceroyl-CoA, which reacted somewhat better with dihydrosphingosine than with sphingosine. The keto analogue of dihydrosphingosine acted as an acceptor, with stearoyl-CoA, to form “keto- ceramide.”

The microsomal preparation also converted the acyl-CoAs to free fatty acid and polar lipid during the incubations. Ceramide synthesis was found to occur primarily in micro- somal membranes, but ceramide hydrolase was rather widely distributed in subcellular fractions; its highest specific activity was in the lysosome-rich fractions. Free stearic acid did not react to form ceramide, yet it could react with ethanol- amine to form the analogous amide. From these and re- lated considerations, we conclude that the synthetic capa- bility of ceramide hydrolase has little or no physiological significance.

Ceramidei is known to be a precursor of most or all sphingo-

* This study was supported in part by Research Grant NS-03192 from the United States Public Health Service.

$ Postdoctoral trainee, supported by Training Grant MH-07417 from the National Institute of Mental Health, United States Public Health Service. Present address, Department of Neurol- ogy, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461.

1 Ceramide is the N-acyl derivative of a long chain base. Un- less otherwise indicated, the term is restricted in this paper to N-acyl groups in which the fatty acid is not hydroxylated.

lipids. Ceramide containing nonhydroxy fatty acid is a pre- cursor of ganglioside (1) and sphingomyelin (24) while ceramide containing hydroxy fatty acid serves as acceptor for galactose in forming hydroxy cerebroside (5). The biosynthesis of cer- amide was demonstrated by Sribney (6) to occur in microsomal preparations from rat brain and liver by an acyltransferase reaction.

Fatty acyl-CoA + long chain base + ceramide + CoA

Yavin and Gatt (7) purified an enzyme (“ceramidase”) from rat brain which catalyzes the hydrolysis of ceramide, as well as its formation, by the reaction

Fatty acid + long chain base & ceramide + H20

They suggested that this enzyme is responsible for the synthesis of ceramide in viva and that Sribney’s system might actually operate through the mediation of a thiolesterase, which would release free fatty acid from the acyl-CoA and allow ceramidase to carry out the synthetic reaction.

To help clarify the matter, we have characterized the brain system which is active in the synthesis of ceramide from long chain base and acyl-CoA. Our studies support the hypothesis of Sribney that acyl-CoA is the physiological substrate in cer- amide formation.

The ceramide portion of complex sphingolipids isolated from brain is quite heterogeneous, differing in chain length and degree of saturation of both the amine and fatty acid moieties (8-10). We have compared four fatty acyl-CoA donors in the ceramide synthesizing system and conclude that the distribution of N-acyl fatty acids is controlled, at least in part, by specificity of the acyltransferase.

EXPERIMENTAL PROCEDURE

Materials-nn-Erythro-sphingosine and nn-erythro-dihydro- sphingosine were synthetic products obtained from Miles-Yeda, Ltd., via Miles Laboratories, Elkhart, Indiana. The sphingo- sine was heavily contaminated with dihydrosphingosine and was purified by preparative thin layer chromatography. DL-

Three-sphingosine was a gift from Dr. Paul W. O’Connell of the Upjohn Company, Kalamazoo, Michigan. A sample of i4C-3-ketodihydrosphingosine was a gift from Drs. Robert Brady and Esmond Snell. Nonradioactive fatty acids and acyl chlorides were obtained from the Hormel Institute, Austin, Minnesota. Carboxyl-labeled fatty acids, palmitic, stearic,

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and oleic, were obtained from Calbiochem, as was the CoA. Detergents used included Tween 20 (Rohm and Haas), Myrj 59 (formerly G2159, from Atlas Chemical Industries), Triton X-100 (Rohm and Haas), and Miranol L2M (a gift from Miranol Chemical Company, Irvington, New Jersey). The first three detergents are nonionic while the last is zwitterionic. l-14C- Lignoceric acid was available in this laboratory (11). The 3-keto analogue of stearoyldihydrosphingosine was made by oxidation with dichromate (12).

Analytical grade Celite, a purified diatomaceous earth, was a Johns-Manville product. Swiss-Webster white mice (CFW) were obtained from Carworth, Portage, Michigan.

Analytical Methods-Thin layer chromatography was done on 0.5-mm layers of Silica Gel G (Merck 7731) unless otherwise specified. Purity of the bases used as substrates was determined by thin layer chromatography with chloroform-methanol-water- ammonia (280:70:6:1) (13). In the assay for ceramide forma- tion, separation of ceramide from free fatty acid and “polar lipid” was accomplished by thin layer chromatography with chloroform-acetic acid (9: 1). Ceramide thus isolated was re- covered by elution of the silica gel in a small column with chloro- form-methanol-water (7 :7 : 1) (14). N-Acylsphingosine sepa- rated clearly from the corresponding N-acyldihydrosphingosine by chromatography on borate-impregnated plates (15). Lipids were visualized by exposure to iodine vapor, except where indi- cated. It was noticed that saturated ceramide spots were rela- tively nonreactive to iodine, compared with acylsphingosines, while bromthymol blue gave more equal darkening.

Reversed phase thin layer chromatography of methyl esters of fatty acids worked well with plates impregnated with mineral oil and developed with nitromethane-acetonitrile-acetic acid (75:lO:l) (16). The esters were detected with a hydroxyl- amine-ferric chloride spraying procedure (Spray 69 as described by Waldi (17)). Phospholipids were separated on silica gel plates made with 10 mM Na2C03 (18) and visualized with the Dittmer-Lester phosphate spray (19). Fatty acyl hydroxamates were separated on precoated silica gel plates (Brinkmann 5763) with toluene-methanol (8 :2) (20). Free fatty acid was sepa- rated from other lipids by thin layer chromatography with hexane-ether-acetic acid (85: 15 :4). Ascending chromatography of acyl-CoAs was carried out on Paper 2043-B of Carl Schleicher and Schuell Company with butanol-acetic acid-water (5 : 2 : 3) and isopropyl alcohol-pyridine-water (1: 1: 1) (21).

The fatty acids of ceramide were released by methanolysis in O-ring sealed tubes (22) and characterized by gas chromatography with Kovar columns (23). Evaporations with a stream of nitrogen were carried out with the N-EVAP (Organomation Associates, Shrewsbury, Massachusetts). Filtration of incuba- tion mixtures was performed with pressure funnels, which were made by sealing a 28/15 ball joint to the top of a sintered glass funnel. Radioautogram and counting procedures are described in the accompanying paper (15).

Preparation of Substrates and Stundards-Mixed ceramides containing both nonhydroxy and hydroxy fatty acids were pre- pared from bovine spinal cord cerebroside by the procedure of Carter, Rothfus, and Gigg (24). Stearoyl-nn-sphingosine and stearoyl-nn-dihydrosphingosine were synthesized from stearoyl- chloride and base by reaction in sodium acetate and aqueous tetrahydrofuran (3). Radioactive palmitoyl-, stearoyl- and oleoyl-CoAs (about 10 mCi per mmole) were made by the enzymatic procedure of Kornberg and Pricer (21). The product

was isolated by perchloric acid precipitation and elution from a glass wool column as detailed by Galliard and Stumpf (25). Lignoceroyl-CoA was prepared by the mixed anhydride method, as described by Young and Lynen (26), with several modifica- tions because of the high insolubility of the long chain acid. Labeled lignoceric acid (60 PC in 30 pmoles) was dissolved in 2 ml of tetrahydrofuran (distilled from KOH + NaPb alloy) and 0.4 ml of chloroform. The reaction with ethyl chloroformate was carried out at O”, instead of -15”. After the reaction with coenzyme A (30 pmoles) the unreacted lignoceric acid was re- moved with five washes of hexane-benzene (1 :I). This wash- ing did not remove all the free acid, and possibly a more polar solvent should be used. The yield of lignoceroyl-CoA was over

40%. Examination of the labeled palmitoyl-, stearoyl-, and oleoyl-

CoAs by paper chromatography with two systems revealed a major spot containing over 95% of the radioactivity; some trail- ing was noticeable in each lane. Lignoceroyl-CoA did not mi- grate. All four acyl-CoAs were converted to acylhydroxamates by incubating 50 nmoles of acyl-CoA in 0.2 ml of 1 M neutralized hydroxylamine containing 0.2 M Tris-HCI, pH 7.4, for 60 min at 37”. The incubation mixtures were washed by partitioning (27), and the hydroxamates were characterized by thin layer chromatography and radioautography. It was found that the medium chain acyl-CoAs were contaminated by less than 1% free fatty acid while lignoceroyl-CoA contained about 10% free acid.

Nonradioactive acyl-CoAs, prepared by the Seubert (28) or mixed anhydride method (26), were used to adjust the specific activities of the radioactive compounds.

Preparation of Microsomes-Mice (16 days old) were decapi- tated, and the brains were removed and homogenized with 4 volumes of 0.25 M sucrose. A glass homogenizer with me- chanically driven Teflon pestle was used. After addition of 3 more volumes of sucrose solution the homogenate was spun at 20,000 x g for 20 min. The resultant pellet was resuspended in 4 volumes of sucrose and centrifuged for 25 min at 20,000 x g. Combination of the supernatants and centrifugation for 90 min at 55,000 x g yielded a microsomal pellet which was resuspended in sucrose to a final volume of 0.6 ml per g of brain. The suspen- sion was stored as long as 4 weeks at -18” before use.

Subcellular fractionation was carried out as described by De Robertis et al. (29).

Assay for Ceramide Bioqnthesis-The usual incubation mix- ture consisted of 0.25 mg of long chain base, 25 pmoles of potas- sium phosphate buffer, 0.5 pmole of dithiothreitol, 1 pmole of ATP, 0.2 ml of microsomes (3.5 to 4 mg of protein), and 0.16 pmole of l4C-acyl-CoA (added last) in a total volume of 0.5 ml. The amine was coated on 25 mg of Celite by pipetting a suspen- sion of Celite in chloroform into the incubation tube, then a solution of base in chloroform-methanol, and then evaporating the mixture to dryness with a stream of nitrogen. The ATP was neutralized before use, and the buffer was added as a 0.5 M solution, pH 7.4 at 25”. Incubation was carried out at 34’ with violent agitation for 10 min and terminated by the addition of 8.5 ml of chloroform-methanol (2: 1).

We removed the protein and Celite by filtration, rinsed the incubation tube and funnel with 1 ml of chloroform-methanol (2:1), and added 2 ml of 2 M KC1 to the filtrate. The resultant layers were separated by centrifugation and the lower layer was washed with 5 ml of water-methanol (1:l) containing 0.5 M

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344 SpeciJicity in Ceramicle Biosynthesis by Brain Vol. 245, Ko. 2

FIG. 1 (zqper). ltadioautogram of t,)tal lipids extracted after incubation of microsomes and a long chain base with radioactive palmitoyl- or stearoyl-CoA. Incubation conditions are as de- scribed under “Experimental Procedure” except that incubation time was 15 min. Lipids were isolated, mixed ceramide was added as carrier, and they were separated by thin layer chromatography in chloroform-acetic acid (9: 1). The stearoyl-CoA (1,250 cpm per nmole) and palmitoyl-CoA (11,000 cpm per nmole) were used at different specific activities in order t.o cause similar darkening of the ceramide region. Stippled lines represent lipids visible with iodine and were traced directly ont,o the x-ray film with the azo print of the thin layer plate as a guide. The acceptor amine and radioactive acyl-CoA are, respectively, Lane 1, dihydrosphingo- sine and palmitoyl-CoA; 2, sphingosine and palmitoyl-CoA; 6, dihydrosphingosine and stearoyl-CoA; 7, sphingosine and stearoyl- CoA. Standards are: 3, synthetic stearoyl sphingosine; 4, syn- thetic stearoyl dihydrosphingosine; 5, mixed ceramides. Acyl groups are identified by carbon nllmber and number of double bonds; D is dihydrosphingosine; S is sphingosine; PL is polar lipid; FA is fatty acid (e.g. 16:0-D is palmitoyldihydrosphingo- sine).

FIG. 2 (lower). ltadioautogram of purified palmitoyl and ste- aroyl ceramides. The radioactive ceramide bands from a plate similar to that shown in Fig. 1 were scraped and elut,ed. Stearoyl-

KC1 and then with 5 ml of water-methanol (1: 1). After evapo- rating the lower phase to dryness with nitrogen, we added 0.1 mg of mixed ceramides as carrier and separated the lipids by thin layer chromatography. After drying and exposure to iodine vapor, the plates were photographed with an overhead projector as light source and azo blueprint paper (30) and then exposed to x-ray film, and the labeled spots were scraped off.

Control tests with labeled stearoylsphingosine yielded com- plete recovery in the ceramide spot after treatment with the partitioning and chromatographic steps. The medium chain acyl-CoAs were removed entirely from the lower layer by the washing procedure, but up to 20% of the lignoceroyl-Cob re- mained in the lower layer. This compound migrated with the polar lipids on thin layer plates.

In preliminary experiments on the isolation of the ceramide from incubations in vitro we used 1 M KOH prior to removal of unreacted acyl-CoA. This resulted in a large amount of arti- factual labeled ceramide, evidently the result of ammonolytic reaction with the long chain base.

Assay for Ceramide Hydrolysis-Ceramidase activity was de- termined by incubating an emulsion of i4C-stearoylsphingosine at pH 5.2 for 180 min (31, 32). The total lipids were extracted and washed as in the acyltransferase assay described above, and the liberated i4C-stearic acid was isolated by thin layer chro- matography and counted.

Assay for Stearoylethanolamide Synthesis-Enzymatic synthe- sis of the stearoyl amide of ethanolamine, observed by Bachur and Udenfriend (33)) was assayed as in the acyltransferase assay procedure, with substitution of 0.16 M ethanolamine for the long chain base and the potassium salt of l*C-stearic acid (50 nC in 50 nmoles) for the acyl-CoA. Incubation was for 45 min.

RESULTS

Validity of Assay Procedure and Characterization of Products- In preliminary experiments the mixed ceramides used as chro-

matographic standards were subjected to preparative thin layer

chromatography with chloroform-acetic acid (9: 1). The upper band, containing the nonhydroxy ceramides, separated into two poorly demarcated bands, the lower of which was much less intense (see Lane 5 in Fig. 1). The ceramides in the two zones were analyzed separately by methanolysis and gas chromatog- raphy. As expected from the known ability of silica gel to separate fatty acid derivatives according to the acid chain length, we found the upper band to contain primarily lignocerate and

nervonate in the N-acyl position and the lower band to contain primarily stearate. The two bands are designated as “long chain ceramide” and “medium chain cersmide.”

Fig. 1 illustrates the separation of labeled ceramides from other radioactive lipids formed in the incubation system. Stearoyl- sphingosine cochromatographs with the medium chain ceramide

of the mixed ceramides used as carrier (Lane 7) and biosynthetic palmitoylsphingosine migrates distinctly more slowly (Lane 2).

dihydrosphingosine (upper spot) and stearoylsphingosine (lower spot) were added as carrier to theextractedlipids beforerechroma- tography on borate-impregnated silica gel with chloroform-meth- anol (9: 1). Radioactive ceramides are, respectively, Lane 1, pal- mitoyldihydrosphingosine; 2, palmitoylsphingosine; 6, stearoyl- dihydrosphingasine; 7, stearoylsphingosine. Standards are: 3, stearoylsphingosine; 4, stearoyldihydrosphingosine; 5, mixed ceramides (upper spot contains long chain N-acylsphingosine, middle spot is primarily stearoylsphingosine, and the lower spot has hydroxy fatty acids in the N-acyl position).

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The acyldihydrosphingosines chromatograph ahead of their sphingosine analogues in this system. An unknown radioactive material (Band X) appears in incubations made with labeled palmitoyl-CoA but not when a TPNH-generating system (15) is included in the incubation mixture.

To characterize the biosynthetic ceramides further, the cera- mide regions from plates like that shown in Fig. 1 were eluted and the lipids were rechromatographed on borate plates (Fig. 2). It is evident that acylsphingosine is formed when sphingo- sine is the acceptor base incubated and acyldihydrosphingosine is formed when dihydrosphingosine is the acceptor. This chro- matographic system also indicates the purity of the radioactive ceramide isolated from the first chromatographic system (Fig. 1) and validates the isolation procedure used in the assay.

The assay for oleoyl and lignoceroyl ceramides, as well as their further characterization on borate plates, is shown in Figs. 3 and 4. The oleoyl ceramides move slightly ahead of the stearoyl ceramides added as carriers, while the lignoceroyl ceramides migrate with the long chain ceramides used as carrier in these lanes. The long chain amides of dihydrosphingosine in the carrier were not visible because of the relatively small amount present.

To show that the fatty acyl group added to the incubation mixture was not modified before incorporation into the ceramide, we cleaved the different ceramides (isolated by chromatography in chloroform-acetic acid) by acid methanolysis. Extraction of the methanolysate with hexane yielded the fatty acid methyl ester fraction, which contained about 70% of the radioactivity present in the oleoyl ceramide, and over 95% of the activity in the other ceramides. The methyl esters thus obtained were chromatographed in a reversed phase system with carriers. Radioautography showed that all the radioactivity was in the expected ester bands. The RFs of the esters were 0.06 (ligno- cerate), 0.22 (stearate), 0.33 (palmitate), and 0.36 (oleate).

In additional experiments, the lipids obtained by incubation of the ceramide synthesizing system with labeled acyl-CoAs were treated with alkali to cleave ester-type lipids and then processed through the standard isolation and counting pro- cedure. The ceramides isolated in this way and in the usual way were found to have the same amount of radioactivity. From this it is seen that ester-bound fatty acids do not contaminate the ceramides isolated in the standard procedure.

Examination of the polar lipids obtained as a by-product in the incubation of the medium chain acyl-CoAs showed the presence of 1% in the bands corresponding to phosphatidylcholine, serine, and -ethanolamine. As will be indicated later, stearoyl-CoA was much more effectively incorporated into the polar lipid fraction than either palmitoyl- or oleoyl-CoA. This differential increase in incorporation of radioactivity was due primarily to formation of a lipid that migrated in the region of phosphatidyl ethanolamine.

Requirements of Synthetic Reaction-Ceramide synthesis does not seem to require a metal cofactor, ATP, or thiol protecting agent (Table I). Tris buffer was found to be distinctly inferior to phosphate buffer. The addition of various detergents failed to stimulate the formation of stearoyldihydrosphingosine, even when Celite was not used to help disperse the base (Table II).

In experiments on the biosynthesis of cerebroside (5), we found considerable dependence on the dispersal of the lipoidal sub- strate on Celite. In the present study (see Fig. 7 below), the use of Celite produced roughly a doubling of activity.

Under conditions similar to the assay for ceramide synthesis, free 14C-stearic acid was not incorporated into ceramide. The acid was added as the ammonium salt, as a suspension in the presence of various detergents, or coated on Celite. To show that free stearic acid could be enzymatically converted to an amide in our system, we incubated the labeled acid with ethanol- amine (see “Experimental Procedure”). Approximately 6

FIG. 3 (upper). Radioautogram of total lipids extracted after incubation of microsomes and a long chain base with radioactive oleoyl- or 1ignoceroyLCoA. Incubation conditions and separation of radioactive lipids as in Fig. 1, except t’hat the radioactive pre- cursors were oleoyl-CoA (7,000 cpm per nmole) and lignoceroyl- CoA (2,500 cpm per nmole). The acceptor and radioactive acyl- CoA are, respectively, Lane 1, sphingosine and oleoyl-CoA; 2, dihydrosphingosine and oleoyl-CoA; 5, sphingosine and lignocer- oyl-CoA; 6, dihydrosphingosine and lignoceroyl-CoA. Standards are: 3, stearoylsphingosine; 4, stearoyldihydrosphingosine; 5, mixed ceramides.

FIG. 4 (lower). Radioautogram of purified oleoyl and ligno- ceroyl ceramides. Samples prepared as in Fig. 2. Stearoyldihy- drosphingosine and stearoylsphingosine were added as carrier to the radioactive oleoyl ceramide while mixed ceramides were added as carrier for the radioactive lignoceroyl ceramide. R.adioactive ceramides are, respectively, Lane 1, oleoyldihydrosphingosine; 2, oleoylsphingosine; 6, lignoceroyldihydrosphingosine; i’, lignocer- oylsphingosine. Standards are: 3, stearoyldihydrosphingosine; 4, stearoylsphingosine; 5, mixed ceramides.

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346 XpeciJicity in Ceramide Biosynthesis by Brain Vol. 245, No. 2

nmoles of stearoylethanolamide were formed, a conversion efficiency of 12%.

Time Course of Ccramide Formation-Fig. 5a shows the forma- tion of acyldihydrosphingosine from labeled acyl-CoAs as a

TABLE I

E$ect of cofactors on ceramide synthesis

Incubation tubes contained 0.25 mg of dihydrosphingosine on 25 mg of Celite, 25 pmoles 0.05 M potassium phosphate buffer, pH 7.4, 3.8 mg of microsomal protein, 160 nmoles of W-stearoyl- CoA (3,100 dpm per nmole), and additions as listed below in a total volume of 0.5 ml.

Addition Cer-

amide 1 ‘OlTllfX

Nothing 1 InM EDTA 1 mivr EDTA, 2 mM

MgCL 1 mM MgClz

2 mM MgCl,

4 InM MgClt 1 IIIM MnClz

2 mM MnClz

wwles 14.8 14.6 12.0

11.6

9.6

9.4 15.2

16.0

Addition

1 mM CaClz 2 mM ATP 1 mu dithiothreitol

2 mM ATP, 1 mM dithio- threitol

2 mM MgC12, 1 mM EDTA, 2 mM ATP, 1 mM dithio- threitol

2 mM ATP, 1 mM dithiothre- itol, - phosphate + 0.05 M Tris, pH 7.4

Cer- amide tormed

lwmles 12.4 14.4 15.2

14.4

16.6

9.6

-

TABLE II Effect of detergents on ceramide synthesis

Incubations conducted as described in “Experimental Pro- cedure” with the following additions of ‘detergents. Results are tabulated as nanomoles of 14C-stearoyl-CoA (3,100 dpm per nmole) converted to stearoyldihydrosphingosine.

Addition

Nothing Tween 20, 0.05% Tween 20, 0.10%

Tween 20, 0.2070 - Celite + Tween 20,

0.05% - Celite + Tween 20,

0.10% - Celite + Tween 20,

0.2o$g

Cer- amide tormw

nmoze. 16.4 16.0 12.0

9.6 12.6

11.6

9.6

Addition

Miranol L2M, 0.1% Sodium cholate, 0.1% Sodium taurocholate,

0.1% Triton X-100, 0.1% G2159, 0.1%

-

f _-

-

Cer- amide ormed

?PW&Ol~S

15.2 1.8 2.8

3.5 16.8

FIG. 5. Time course of conversion of radioactive acyl-CoA to ceramide, polar lipid, and free fatty acid. Incubation conditions as described under “Experimental Procedure” except that time was varied as indicated. The acceptor was dihydrosphingosine, and specific activity of each radioactive acyl-CoA was 1,250 cpm per nmole. Total lipids were isolated and separated by thin layer chromatography. After radioautography, regions containing radioactive ceramide, polar lipid, or free fatty acid were scraped and counted. Results: a, ceramide formed; b, polar lipid formed; c, free fatty acid released. Curves are identified as explained in Fig. 1.

function of time. A logarithmic scale for the ordinate has been used to cover the 50-fold difference between the extent of forma- tion of oleoyl- and stearoyldihydrosphingosine. In each case the amount of ceramide formed was somewhat proportional to time for the first 15 min, and synthesis tis ended within ap-

I-

/ -I

I 0

I I , I I I I I c

i-

16~0~D

I I I I I I 1 I I

IO 20 30 40 50 60 70 80 90

Time (min)

IO 20 30 40 50 60 70 80 90

Time (min)

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Issue of January 25, 1970 P. Morel1 and N. 8. Radin 347

proximately 25 min. Almost 20% of the stearoyl-Coil was converted to ceramide by this time.

The time course of release of free fatty acid (Fig. 5b) and of formation of polar lipids (Fig. 5~) is also shown. Conversion of lignoceroyl-CoA is not shown in Figs. 5b and 5c because con- tamination of the starting material with free fatty acid and carryover of unreacted lignoceroyl-Cob through the extraction procedure interfered with accurate determination of the fatty acid and polar lipid fractions. As with the ceramides, the forma- tion of fatty acid and polar lipids was ended by about 25 min in each case. While this simultaneity could be the result of simul- taneous deterioration of the several enzymes involved in the synthetic and hydrolytic reactions, it is much more likely that all the available acyl-CoAs were hydrolyzed by the end of 25 min.

Palmitoyl- and oleoyl-CoA were hydrolyzed to fatty acids more rapidly and to a greater extent than stearoyl-CoA. The converse was observed for the formation of ceramide and polar lipid. The sum of the radioactivity in each of the lipids formed (including estimates for the minor radioactive bands) corresponds to about 110 nmoles for each of the three medium chain precur- sors. The remaining 50 nmoles of acyl-CoA were presumably unavailable for reaction, bound to either protein or long chain base (see below).

Dependence of Ceramide Formation on Acyl-CoA Concentration -The dependence of ceramide formation on concentration of acyl-CoA is shown in Fig. 6, a to d. An acyl-CoA concentration of about 0.5 mM is near optimal for formation of stearoyl and lignoceroyl ceramides, the latter showing somewhat more sensi- tivity to high levels of acyl-CoA. As indicated in Fig. 5, these two are formed to a much greater extent than the other two ceramides. This preferential synthesis is seen over a wide range of acyl-CoA concentrations (Fig. 6).

There was little difference between sphingosine and dihydro-

“k-ACYL-CoA (mM)

sphingosine as acceptors in the case of the palmitoyl- and oleoyl- CoAs, but the stearate reaction proceeded somewhat better with sphingosine, and the lignocerate with dihydrosphingosine. In other comparisons, threo-sphingosine was found to be utilized as efficiently as the erythro analogue with palmitoyl- and stearoyl- CoA as acyl donor. The 3-keto analogue of dihydrosphingosine proved to be a fairly good acceptor for the stearoyl moiety, 7% (6 nmoles) of the keto base being converted to keto ceramide. The radioactive product cochromatographed with the synthetic ketoceramide (borate plate, chloroform-methanol (95: 5)). While other workers have shown similar low specificity in the utilization of normal and abnormal bases for other sphingolipid reactions (34-37), the ability of the keto base to act as an ac- ceptor raises the interesting possibility of an alternate route to ceramide biosynthesis, namely the formation of ketoceramide and reduction by TPNH.

Fig. 6e shows that stearoyl-CoA is more effective than either palmitoyl- or oleoyl-CoA for the formation of polar lipids, at all concentrations tested. Fig. 6f shows that the thiolesterases in our microsomes are far from saturated at the levels of acyl-CoA used.

Dependence of Ceramide Formation on Amount of Base Present as Acceptor-Fig. 7 shows a broad optimal range (0.15 to 0.4 mg) for the amount of base in the standard incubation system, regardless of which base or acyl-CoA is present. The relative efficiencies of sphingosine and dihydrosphingosine are seen to be similar to those noted in Fig. 6.

The decreased synthesis of ceramide at high levels of amine seen in Fig. 7, a and b, may be due to binding of part of the acyl- CoA. That such binding can occur was shown by incubating labeled stearoyl-CoA with base under conditions similar to that used in the acyl transferase assay, except for the omission of

4- ld) 24:0-D

14C-ACYL-CoA (mM)

160 - (f)

“C-ACYL-CoA (mM1

FIG. 6. Conversion of radioactive acyl-CoAs to ceramide, polar stearoyl; c, oleoyl; cl, lignoceroyl. Since each concentration point lipid, and free fatty acid as a function of acyl-CoA concentration. of a particular acyl-CoA was determined twice (once with sphingo- Incubation conditions as described under “Experimental Pro- sine and once with dihydrosphingosine as acceptor) the experi- cedure” except that the concentration of acyl-CoA (1,250 cpm per mental points indicated for e, polar lipid formation, and f, free nmole) was varied as indicated. The acceptor was either sphingo- fatty acid released, are the average of two similar values. sine or dihydrosphingosine. Ceramides formed: a, palmitoyl; b,

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348 Specifiity in Ceramide Biosynthesis by Brain Vol. 245, No. 2

enzyme. Sphingosine and dihydrosphingosine both precipitated all the acyl-CoA, undoubtedly as a salt.

Dependence of Ceramide Formation on Enzyme Concentration- The amount of ceramide formed in a lo-min incubation increases with the amount of microsomes (Fig. S), but unexpectedly little activity is seen below a level of 1 mg of protein. Probably this reflects the sensitivity of the acyltransferase to the detergent action of acyl-CoAs at low membrane to acyl-CoA ratios.

01 1 I I 1 I 1 0.1 0.2 0.3 0.4 0.5 0.E

Long Chain Bose (mg)

FIG. 7. Conversion of radioactive acyl-CoAs to ceramide as a fun&ion of the amount, of base present. Conditions as described under “Experimental Procedure” (acyl-CoAs at 1,250 cpm per nmole) except that the amount of base per tube was varied as in- dicated. The ratio of base to Celite was constant (1:lOO). Cer- amides formed: a, palmitoyl; b, stearoyl; c, lignoceroyl. X indi- cates incorporation into ceramide when dihydrosphingosine was the acceptor in the absence of Celite.

Polar lipid formation also increases as the microsomal con- centration is raised. This is probably a reflection of the increas- ing amount of endogenous acceptors being added with the micro- somes. The increases in amount of ceramide and polar lipid formation take place at the expense of the competing thiolesterase reaction.

The formation of lignoceroyldihydrosphingosine is dependent on enzyme concentration in a similar manner, except that it plateaus completely at protein levels above 5 mg per tube.

Localization of Acyl-CoA: Long Chain Base Acyltransferase and Ceramidase Activities-A comparison of various subcellular brain fractions (Table III) shows that the bulk of the synthetic activity is present in the microsomal fraction, which also has by far the highest specific activity. Some activity in the myelin fraction may be due to contamination with microsomal material. The assays for ceramide formation were done at two levels of protein concentration and showed relatively good proportionality to enzyme concentration. A separat,e experiment indicated that

Protein (mg)

FIG. 8. Conversion of stearoyl-CoA to ceramide, polar lipid and free fatty acid as a function of protein concentration. Condi tions as described under “Experimental Procedure” (stearoyl CoA at, 1,250 cpm per nmole), with dihydrosphingosine as acceptor except, that the amount of microsomal protein per tube was varied as indicated.

TABLE III Subcellular distribution of ceramide synthesis and hydrolase activities

Activities in the synthetic system are derived from IO-min incubations; those in the hydrolytic system from 180.min incubations. Hydrolytic incubations contained 0.3 nmole of W-stearoyl sphingosine (56,000 cpm) emulsified in a mixture of detergents (32).

Brain fractiona Protein in assay tube

Nuclei and debris. ....... 1.0 Microsomes. ............. 2.5 (A) Myelin. ............. 3.2 (B) Nerve endings. ...... 0.8 (C) Nerve endings. ...... 1.7 (D) Lysosomes ........... 1.2 (E) Mitochondria ........ 0.9

Synthesis of ceramide

Stearoyldibydrosphingosine Lignoceroyldihydrosphingosine

W%Olt?S/WZg moles/bra&z Tmoles/mg protein protein mmlcs/brain

0 0 0.05 1.5 3.0 48 0.60 9.4 0.18 3.4 0.01 0.1 0.5 2.3 0 0 0.36 3.4 0.02 0.2 0.1 0.7 0 0 0.18 0.9 0 0

Ceramidase activity

c@n fatty acid/mg @m fatty acid/brain pot&z equivalent

490 14,000 510 8,000

86 1,600 900 4,200

1,000 9,400 1,800 12,200 3,500 18,000

0 Fractions identified by letters were prepared by the discontinuous density gradient system of De Robertis et al. (29); the labels are nominal.

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Issue of January 25, 1970 P. Morel1 and N. S. Radin 349

utilization of palmitoyl-CoA for ceramide formation followed the in utilization of acyl-CoAs by the transferase is similar to what same distribution pattern. is expected.

The same subcellular preparations were also assayed for ceramidase activity, and the results were tabulated in terms of counts per min of released stearic acid. This mode of presenta- tion was used because the radioactive ceramide used as substrate was at a very high specific activity and was diluted to an un- known extent by endogenous ceramide (either present initially or liberated from complex sphingolipids over the long time period of the assay). All assays were done at two enzyme levels, and there was considerable deviation from linearity in each case; the data from the lower enzyme level were used in making Table III. This deviation was most serious in Fractions D and E, indicating that the most serious underestimation of activity is in these fractions. Despite this complication, Fractions D and E were found to have the highest specific activities for ceramidase. Microsomes show one of the lowest specific activities.

The distribution of ester-linked fatty acids in brain is quite different from that in sphingolipids. In young rats (which in terms of neural development seem to be similar to mice) about 85% of the brain fatty acids are ester-linked and consist pri- marily of palmitate, stearate, oleate, and polyunsaturated acids in roughly equal proportions (45). Since deposition of these fatty acids in ester linkage goes on at roughly similar rates (45) and involves acyl-CoAs, it appears possible that ample concen- trations of these compounds are available to the acyltransferase. If this is true, the selectivity of the enzyme makes it an important controlling factor in sphingolipid biosynthesis.

The heterogeneity of the distribution of ceramidase activity is fairly typical of lysosomal enzymes. Fractions D and E proba- bly contain the highest concentrations of lysosomes.

It is possible that the specificity we observed for ceramide synthesis is the sum of two or more enzymatic activities possess- ing more extreme specificities. The transferase in neurones may accept only medium chain acyl-CoAs, with stearate as the preferred substrate, while glia may contain a similar transferase as well as a transferase accepting only long chain acyl-CoAs. Such a duality could explain the curious relative lack of inter- mediate chain acids (Clezz) in sphingolipids.

DISCUSSION

Physiological Signijicance of Acylimnsferase SpeciJicity- Analytical work from many laboratories indicates that the fatty acid distribution in brain sphinqolipids depends on the type of sphingolipid, the age of the animal, and its location in brain. Sphingomyelin of white matter contains both long chain and medium chain acids, while gray matter sphingomyelin contains mostly stearic acid (3840). Cerebrosides contain mainly long chain acids, both hydroxy and nonhydroxy, with some skewing of the distribution toward shorter acids in some areas (38, 41). While some stearate occurs in cerebrosides, hydroxystearate is quite rare. Gangliosides contain primarily medium chain acids (42, 43). Free ceramides occur in brain at very low concentra- tions (44); the major part of the fatty acids are medium chain. It is not known whether this pool of ceramide is the result of synthetic or degradative processes, or both. Palmitate and oleate are relatively rare in all brain sphingolipids.

The interpretation of enzyme data derived from acyl-CoAs as substrate has been coming under increasingly sophisticated scrutiny. It is evident that fatty acyl-CoAs combine rather firmly with microsomal protein, and it is possible that at higher protein to acyl-CoA ratios much of the substrate may be less available for enzyme action. This explains the decrease in slope of the ceramide synthesis-microsome concentration curve (Fig. 8) and a similar observation by Abou-Issa and Cleland (46). At low protein to acyl-CoA ratios the unexpectedly low rate of synthesis may be due to distortion of the enzyme by nonspecifi- tally bound acyl-CoA as well as emulsification of the ordered membrane structure. These interfering effects may occur to different degrees with different acyl-CoAs. Such complications emphasize the necessity of comparing various acyl-CoAs under a variety of conditions.

Since ceramide appears to be an intermediate in the biosyn- thesis of most or all sphingolipids, such biases in the distribution of N-acyl fatty acids could arise by control of the fatty acids available for ceramide formation (e.g. neurones may not synthe- size long chain saturated acids) or the acyl-CoA: long chain base acyltransferase could exercise specificity with respect to the acyl-CoA utilized. It is also possible that both factors operate: the acyltransferase in gray matter may have to select against oleoyl-CoA but not against lignoceroyl-CoA, which may not be formed in gray matter.

A similar care is needed in interpretation of enzyme data ob- tained with long chain bases, since these have the ability to bind anionic lipids in insoluble form, as illustrated by our experiment with labeled stearoyl-Coil. Thus, it is important to determine whether specificity comparisons are made at optimal amine concentrations.

The data in this paper indicate that the ceramide synthesizing system plays an important role in determining the fatty acid distribution pattern. Over a broad range of conditions, the efficiency of utilization of stearoyl, lignoceroyl, palmitoyl, and oleoyl coenzyme As in forming ceramide was found to be in the ratio of about 60:12:3:1. This is quite similar to the distribu- tion ratio of these fatty acids in amide linkage observed in mam- malian brain. The distribution ratio cannot be used to predict accurately the specificity in vitro of the transferase system, be- cause of such additional factors as relative turnover rates and concentrations of individual sphingolipids and acyl-CoAs. Nevertheless, it seems clear that the relative order of efficiency

Possible Role of Ceramidase in Ceramide Biosynthesis-Yavin and Gatt have suggested (7) that the long chain base acyltrans- ferase might not exist and that the acyl-CoA is actually hy- drolyzed to free fatty acid, which is then converted by ceramidase to ceramide. However, Sribney found that free palmitic acid did not react with sphingosine unless CoA and ATP were present (6). We found no ceramide synthesis from stearic acid in our system, yet our preparation actively formed an analogous com- pound, stearoylethanolamide, from stearic acid and ethanol- amine. Moreover, our time course study (Fig. 5) showed that cessation of ceramide synthesis occurred at the point of maximal formation of free fatty acid. Particularly convincing are the data from the subcellular fractionation experiment (Table III) which showed that the enzymes forming and hydrolyzing ceramide are distributed within the cell very differently.

In contrast to the physiologically normal specificity of the acyltransferase with respect to fatty acid, the synthetic activity of ceramidase is most effective with fatty acids which occur in sphingolipids only to a negligible extent. These are acids which

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350 Specificity in Ceramide Biosynthesis by Brain Vol. 245, No. 2

are relatively water-soluble (oleate, dodecanoate), and it is likely that the synthetic capability of ceramidase depends simply on the driving power of precipitation. The enzyme catalyzes the conversion of a relatively soluble fatty acid to a highly in- soluble ceramide.

Aclenowledgments-We are greatly indebted to Mrs. Carolyn Seidl and Mrs. Inez Mason for their skillful, dedicated assistance. The advice of Dr. Otto 2. Sellinger on subcellular fractionation is greatly appreciated.

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Pierre Morell and Norman S. RadinAcyl Coenzyme A's by Brain Microsomes

Specificity in Ceramide Biosynthesis from Long Chain Bases and Various Fatty

1970, 245:342-350.J. Biol. Chem. 

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