Biochimica et Biophysica Acta, 1116 (1992) 97-103 © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00
97
BBAGEN 23652
A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase Noboru Matsuo, Tomoko Nomura and Genji Imokawa Biological Science Laboratories, Kao Corporation, Tochigi (Japan) (Received 25 October 1991)
Key words: Glucose : ceramide glucosyltransferase; Enzyme assay; Solid phase method
We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransfcrase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction )roduct, [~4C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [laC]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn 2+, Mg 2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Apparent K m values of 8.7 IzM for UDP-glucose and 292 tzM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.
Introduction
Ceramide glucosyltransferase (UDP-glucose:ceramide glucosyltransferase; EC 2.4.1.80) is an enzyme which catalyzes the synthesis of glucosylceramide from ceramide and UDP-glucose. This reaction is the first step in the biosynthesis of gangliosides and other glycolipids [1]. The enzyme is present in chick embryonic brain [2] as well as in mammalian organs such as mouse brain [3], mouse liver [4], porcine submaxillary glands [5] and cultured hamster fibroblast BHK-12 cells [6]. Reactions catalyzed by UDP-glucose:ceramide glucosyltransferase, including those involved in sphingolipid metabolism, have recently been investigated regarding their role in regulation of cell proliferation [7-9] and other cell functions [10-12]. One of the methods most commonly used for measuring this enzyme involves a reaction with micellar ceramide and subsequent solvent extraction of the reaction product, glucosylceramide [16].
Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]l-propanesulfonate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonate; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline; EGTA, ethylene glycol-bis(/3-aminoethylether) N,N,N ', N '-tetraacetic acid. Correspondence: N. Matsuo, Kao Corporation, 2606 Akabane, Ichikaimachi, Haga, Tochigi, 321-34, Japan.
Several attempts have been made to improve assay methods, for example, ceramide adsorbed on celite has been used [13]. Silica gel has also been used as the reaction field for a substrate specificity study in which the reaction takes place on thin layer chromatography silica gel plates [14]. These attempts, however, have not been very successful with regard to improving accuracy and rapidity. The present report describes a simple rapid assay method for determining glucosylceramide formation from UDP-glucose and ceramide immobilized on solid supports. The biological properties of this enzyme have been studied using this new assay method. Materials and methods
Materials Male albino Wistar rats were used as an enzyme source. The animals were 3 - 4 weeks old; substantial amounts of the enzyme have been reported in rats of this age [15]. [~4C]UDP-glucose (11.1 G B q / m m o i ) was purchased from Amersham International (U.K.). Ceramide (type III prepared from bovine brain sphingomyelin, type IV from bovine brain cerebroside), lactosylceramide (prepared from bovine brain), glucosylceramide (prepared from human Gaucher's spleen), galactosylceramide (prepared from bovine brain), UDP-glucose, Mops (3-(N-morpholino)propanesulfonic acid), and CHAPS were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Other detergents
98 were purchased from Boehringer Mannheim (Mannhelm, Germany). Materials for solid support were purchased from Tosoh (Tokyo, Japan). High performance thin layer chromatography (HPTLC) plates were purchased from Merck (Germany). Other reagents were of the highest grade commercially available.
Preparation of enzyme source Rat brains were either frozen in liquid nitrogen and stored at - 8 0 ° C or were used immediately. Brains were minced with scissors and homogenized in 5 vol. of homogenizing buffer (0.25 M sucrose, 1 mM EDTA, 1 mM E G T A , 0.5 mM phenylmethanesulfonyl fluoride, 50 mM Mops, pH 7.4), using a Potter-Elvehjem homogenizer with a mechanically driven teflon pestle at 600 r e v / m i n . The homogenate was fractionated by sequential centrifugation in the same buffer. Since the enzyme activity of the m e m b r a n e fraction obtained by centrifugation at 10000 × g for 30 rain was greater, in terms of both total activity and specific activity, than that obtained by centrifugation at 100000 x g for 60 rain, the former was used to establish the optimal assay condition for the experiment. Preparation of solid phase substrate The ceramide to be used as a substrate was dissolved in a small volume of chloroform. The solid support gel, such as silica gel, was added to the solution. Unless otherwise mentioned, type IV ceramide was used throughout the paper. When type IV ccramide was used, heating of the chloroform solution resulted in a clear solution. After the gel was suspended evenly, chloroform was evaporated under a gentle stream of nitrogen gas. When most of the solvent had evaporated and the gel became white and caked, it was transferred to a glass Petri dish and allowed to dry completely. When silica gel was used, 100 percent of added ceramide was adsorbed to the solid substrate as traced with [~4C]palmytoyl sphingosine. This solid substrate preparation could be stored at - 2 0 ° C for several weeks without any significant loss of its substrate activity. The optimal density of ceramide on the solid support will be discussed in the Results section. Enzyme assay Measurements of the enzyme activity were performed in duplication throughout the paper. The composition of the assay mixture for UDP-glucose:ceramide glucosyltransferase activity was essentially identical to that described by Coste et al. [16], except for the presence of the solid substrate. The reaction mixtures contained m e m b r a n e s (125-500 Izg protein), solid substrate (0.2-20 rag), 5.4 nmol [14C]UDP-glucose (13.9 KBq), 5.5 /,tmol Mops (pH 6.5), 550 nmol MnCI z, 275 nmol MgCI:, 110 nmol N A D H , 550 nmol 2,3-di-
mercaptopropanol, and various concentrations of detergents, in a final volume of 11() #I. This substrate mixture was prepared in required amounts just prior to use and pipetted inlo individual reaction wells of a 96-well micro-titer plate. The top of each well was sealed with a plastic cohesive film (Becton Dickinson, N J, U.S.A.) to aw)id cross contamination and evaporation. During the incubation period, the plate was shaken on a platc mixer to avoid sedimentation of the solid material. The reaction was slartcd by adding 25 p,l of [HC]UDP-glucose; the plates were then incubated at 37°C for 30 min, and the reaction was terminated by diluting the reaction mixture with 200 p.l of cold phosphate-buffered saline (PBS). To remove free [HC] UDP-glucose, the plate was centrifuged (2000 rpm l~r 2 rain) and the resultant pellet was washed hmr times with 200 #1 of PBS. The washed solid substrate was then transferred to scintillation vials and radioactivity was measured by a scintillation counter with Aquazol-2 scintillation fluid (Packard Instruments, U.S.A.). As an alternative assay method, the micellar substrate system described by Coste et al. [5,16] was also used for comparison in certain cases.
Analysis of reaction product The enzyme reaction was carried out with 1 mg silica gel adsorbed with 20 # g of type III ceramide and 212 ~ g of m e m b r a n e protein per assay. After 30 min incubation at 37°C, the reaction product tormed on the surface of the solid support was extracted with chlorof o r m / m e t h a n o l (2: 1, v/v), concentrated under nitrogen gas, and spotted on a T L C plate. The samples containing approx. 10000 dpm were analyzed on silica gel thin layer plates with a solvent system c h l o r o f o r m / m e t h a n o l / w a t e r (65 : 25 : 4, v/v). Borateimpregnated silica gel plate was prepared by immersing silica gel thin layer plates in a 5% solution of sodium tetraborate in methanol for 1 min. Plates were allowed to dry at room temperature and were activated in an oven at 100°C for 2 h before use. The borate impregnated plates were developed in c h l o r o f o r m / m e t h a n o l / w a t e r (100: 300: 4, v/v). The plates were autoradiographed using Kodak diagnostic film X - O M A T AR, and then visualized by treatment with 10% CuSO 4 in 8C/c H3PO 4 solution, followed by heating at 16(t°C for 10 min. Measurement of ceramide adsorbed on silica gel [14C]Ceramide was prepared from [14C]palmitic acid and sphingosine, according to a method reported previously [17]. Ceramide type IV, mixed with [14C]ceramide, was adsorbed to silica-60 gel at a density of 20 ~ t g / m g silica gel by the procedure described above. 1 mg of this radio-labeled solid substrate was incubated in each of the assay mixtures containing various con-
99
centrations of CHAPS in the absence of the enzyme sample. After 30 min incubation at 37°C with shaking, the solid substrate was then washed four times with 200 /xl of PBS. The radioactivity of the silica gel was measured by scintillation counter to estimate the amount of ceramide remaining on the gel.
100
& o z 5O E a~ E
Results
Selection of solid support In order to determine a suitable solid support for the adsorption of ceramide, the enzyme assay was performed in the presence of solid substrates prepared with various possible solid supports as indicated in Fig. 1. In this experiment the detergent CHAPS was provisionally used as a 1% (w/v) final concentration. Of the solid supports tested, TMS-250, Styrene-250, Styrene60, ODS-120A, and ODS-120T have hydrophobie surface properties; the rest of the solid supports tested were hydrophilic. Particle size diameters were 5 /zm except for TMS-250, which had a diameter of 10 /zm [181. As shown in Fig. 1, the amount of glucosylceramide produced on the surface was relatively greater when hydrophilic rather than hydrophobic gel was used, and Silica-60 gel exhibited the highest incorporation of radio-labeled glucose into the ceramide substrate. We therefore chose Silica-60 gel as the solid support for the following experiments.
Effect of ceramide density on [14C]glucose incorporation To evaluate the optimal surface concentration of ceramide, we performed the enzyme assay using ceramide adsorbed to silica gel at densities varying from 0 to 200/zg per mg of silica gel. The detergent CHAPS was again used provisionally at a concentration of 1% 100
8 o
8
c
--~ E
50
N 0
%
% %%
%%
Fig. 1, [14C]Glucose incorporation to ceramide adsorbed on various solid supports. Each solid support was coated with ceramide at a density of 20 p,g of ceramide per mg of solid. 1 mg of these solid substrates and 78 /zg of protein were used per assay. CHAPS concentration was 1% (w/v). Maximum activity corresponded to an activity of 1.54 n m o l / m g of protein per h.
0
I
2
L___
5
10
50
i
_ __
100
Ceramide density (mgceramide/gsilicagel)
Fig. 2. Effects of ceramide density on [t4C]glucose incorporation by rat brain UDP-glucose:ceramide glucosyltransferase. Silica gel was coated with various concentrations of ceramide to yield the ceramide density indicated. 1 mg of each solid substrate was used per assay. The assay conditions were the same as those outlined in Fig. 1. The actual incorporation of glucose at the maximum (100%) was 0.78
nmol/mg membraneprotein per h.
(w/v). Fig. 2 shows that [14C]glucose incorporation increased with increasing ceramide density up to 20 /zg/ml silica gel, reached a plateau, and then decreased. Therefore we fixed the ceramide density at 20 /zg/mg silica gel for the following experiments.
Effects of detergents on [14C]glucose incorporation Fig. 3 shows the effects of various detergents at varying concentrations on [14C]glucose incorporation into ceramide immobilized on a silica gel surface. Whereas the incorporation of radio-labeled glucose to ceramide was reduced by most of the detergents at any concentrations tested, only CHAPS, a zwitterionic detergent, exhibit a significant stimulatory effect at a concentration of 1% (w/v). This characteristic stimulatory and inhibitory profile was also observed with CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-l-propanesulfonate), a zwitterionic detergent similar in structure to CHAPS. When re-measured at a narrower concentration range, the CHAPS concentration achieving maximum incorporation was found to be 1% (w/v) (inset to Fig. 3). Because the reduction of glucose incorporation at higher concentrations of detergents might also result from the release or solubilization of ceramide from the solid support, an alternative assay method previously reported [5,16] was also carried out to assess the effect of detergents on the incorporation of glucose into ceramide. As shown in Table I, the stimulatory and inhibitory pattern observed in this micellar system was similar to that seen in the solid phase procedure. The effect of CHAPS concentration on the release of ceramide from the solid support was further studied using radiolabeled ceramide. [14C]Palmytoyl sphingo-
100 100
........................
•
97
BBAGEN 23652
A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase Noboru Matsuo, Tomoko Nomura and Genji Imokawa Biological Science Laboratories, Kao Corporation, Tochigi (Japan) (Received 25 October 1991)
Key words: Glucose : ceramide glucosyltransferase; Enzyme assay; Solid phase method
We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransfcrase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction )roduct, [~4C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [laC]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn 2+, Mg 2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Apparent K m values of 8.7 IzM for UDP-glucose and 292 tzM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.
Introduction
Ceramide glucosyltransferase (UDP-glucose:ceramide glucosyltransferase; EC 2.4.1.80) is an enzyme which catalyzes the synthesis of glucosylceramide from ceramide and UDP-glucose. This reaction is the first step in the biosynthesis of gangliosides and other glycolipids [1]. The enzyme is present in chick embryonic brain [2] as well as in mammalian organs such as mouse brain [3], mouse liver [4], porcine submaxillary glands [5] and cultured hamster fibroblast BHK-12 cells [6]. Reactions catalyzed by UDP-glucose:ceramide glucosyltransferase, including those involved in sphingolipid metabolism, have recently been investigated regarding their role in regulation of cell proliferation [7-9] and other cell functions [10-12]. One of the methods most commonly used for measuring this enzyme involves a reaction with micellar ceramide and subsequent solvent extraction of the reaction product, glucosylceramide [16].
Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]l-propanesulfonate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonate; Mops, 3-(N-morpholino)propanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline; EGTA, ethylene glycol-bis(/3-aminoethylether) N,N,N ', N '-tetraacetic acid. Correspondence: N. Matsuo, Kao Corporation, 2606 Akabane, Ichikaimachi, Haga, Tochigi, 321-34, Japan.
Several attempts have been made to improve assay methods, for example, ceramide adsorbed on celite has been used [13]. Silica gel has also been used as the reaction field for a substrate specificity study in which the reaction takes place on thin layer chromatography silica gel plates [14]. These attempts, however, have not been very successful with regard to improving accuracy and rapidity. The present report describes a simple rapid assay method for determining glucosylceramide formation from UDP-glucose and ceramide immobilized on solid supports. The biological properties of this enzyme have been studied using this new assay method. Materials and methods
Materials Male albino Wistar rats were used as an enzyme source. The animals were 3 - 4 weeks old; substantial amounts of the enzyme have been reported in rats of this age [15]. [~4C]UDP-glucose (11.1 G B q / m m o i ) was purchased from Amersham International (U.K.). Ceramide (type III prepared from bovine brain sphingomyelin, type IV from bovine brain cerebroside), lactosylceramide (prepared from bovine brain), glucosylceramide (prepared from human Gaucher's spleen), galactosylceramide (prepared from bovine brain), UDP-glucose, Mops (3-(N-morpholino)propanesulfonic acid), and CHAPS were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Other detergents
98 were purchased from Boehringer Mannheim (Mannhelm, Germany). Materials for solid support were purchased from Tosoh (Tokyo, Japan). High performance thin layer chromatography (HPTLC) plates were purchased from Merck (Germany). Other reagents were of the highest grade commercially available.
Preparation of enzyme source Rat brains were either frozen in liquid nitrogen and stored at - 8 0 ° C or were used immediately. Brains were minced with scissors and homogenized in 5 vol. of homogenizing buffer (0.25 M sucrose, 1 mM EDTA, 1 mM E G T A , 0.5 mM phenylmethanesulfonyl fluoride, 50 mM Mops, pH 7.4), using a Potter-Elvehjem homogenizer with a mechanically driven teflon pestle at 600 r e v / m i n . The homogenate was fractionated by sequential centrifugation in the same buffer. Since the enzyme activity of the m e m b r a n e fraction obtained by centrifugation at 10000 × g for 30 rain was greater, in terms of both total activity and specific activity, than that obtained by centrifugation at 100000 x g for 60 rain, the former was used to establish the optimal assay condition for the experiment. Preparation of solid phase substrate The ceramide to be used as a substrate was dissolved in a small volume of chloroform. The solid support gel, such as silica gel, was added to the solution. Unless otherwise mentioned, type IV ceramide was used throughout the paper. When type IV ccramide was used, heating of the chloroform solution resulted in a clear solution. After the gel was suspended evenly, chloroform was evaporated under a gentle stream of nitrogen gas. When most of the solvent had evaporated and the gel became white and caked, it was transferred to a glass Petri dish and allowed to dry completely. When silica gel was used, 100 percent of added ceramide was adsorbed to the solid substrate as traced with [~4C]palmytoyl sphingosine. This solid substrate preparation could be stored at - 2 0 ° C for several weeks without any significant loss of its substrate activity. The optimal density of ceramide on the solid support will be discussed in the Results section. Enzyme assay Measurements of the enzyme activity were performed in duplication throughout the paper. The composition of the assay mixture for UDP-glucose:ceramide glucosyltransferase activity was essentially identical to that described by Coste et al. [16], except for the presence of the solid substrate. The reaction mixtures contained m e m b r a n e s (125-500 Izg protein), solid substrate (0.2-20 rag), 5.4 nmol [14C]UDP-glucose (13.9 KBq), 5.5 /,tmol Mops (pH 6.5), 550 nmol MnCI z, 275 nmol MgCI:, 110 nmol N A D H , 550 nmol 2,3-di-
mercaptopropanol, and various concentrations of detergents, in a final volume of 11() #I. This substrate mixture was prepared in required amounts just prior to use and pipetted inlo individual reaction wells of a 96-well micro-titer plate. The top of each well was sealed with a plastic cohesive film (Becton Dickinson, N J, U.S.A.) to aw)id cross contamination and evaporation. During the incubation period, the plate was shaken on a platc mixer to avoid sedimentation of the solid material. The reaction was slartcd by adding 25 p,l of [HC]UDP-glucose; the plates were then incubated at 37°C for 30 min, and the reaction was terminated by diluting the reaction mixture with 200 p.l of cold phosphate-buffered saline (PBS). To remove free [HC] UDP-glucose, the plate was centrifuged (2000 rpm l~r 2 rain) and the resultant pellet was washed hmr times with 200 #1 of PBS. The washed solid substrate was then transferred to scintillation vials and radioactivity was measured by a scintillation counter with Aquazol-2 scintillation fluid (Packard Instruments, U.S.A.). As an alternative assay method, the micellar substrate system described by Coste et al. [5,16] was also used for comparison in certain cases.
Analysis of reaction product The enzyme reaction was carried out with 1 mg silica gel adsorbed with 20 # g of type III ceramide and 212 ~ g of m e m b r a n e protein per assay. After 30 min incubation at 37°C, the reaction product tormed on the surface of the solid support was extracted with chlorof o r m / m e t h a n o l (2: 1, v/v), concentrated under nitrogen gas, and spotted on a T L C plate. The samples containing approx. 10000 dpm were analyzed on silica gel thin layer plates with a solvent system c h l o r o f o r m / m e t h a n o l / w a t e r (65 : 25 : 4, v/v). Borateimpregnated silica gel plate was prepared by immersing silica gel thin layer plates in a 5% solution of sodium tetraborate in methanol for 1 min. Plates were allowed to dry at room temperature and were activated in an oven at 100°C for 2 h before use. The borate impregnated plates were developed in c h l o r o f o r m / m e t h a n o l / w a t e r (100: 300: 4, v/v). The plates were autoradiographed using Kodak diagnostic film X - O M A T AR, and then visualized by treatment with 10% CuSO 4 in 8C/c H3PO 4 solution, followed by heating at 16(t°C for 10 min. Measurement of ceramide adsorbed on silica gel [14C]Ceramide was prepared from [14C]palmitic acid and sphingosine, according to a method reported previously [17]. Ceramide type IV, mixed with [14C]ceramide, was adsorbed to silica-60 gel at a density of 20 ~ t g / m g silica gel by the procedure described above. 1 mg of this radio-labeled solid substrate was incubated in each of the assay mixtures containing various con-
99
centrations of CHAPS in the absence of the enzyme sample. After 30 min incubation at 37°C with shaking, the solid substrate was then washed four times with 200 /xl of PBS. The radioactivity of the silica gel was measured by scintillation counter to estimate the amount of ceramide remaining on the gel.
100
& o z 5O E a~ E
Results
Selection of solid support In order to determine a suitable solid support for the adsorption of ceramide, the enzyme assay was performed in the presence of solid substrates prepared with various possible solid supports as indicated in Fig. 1. In this experiment the detergent CHAPS was provisionally used as a 1% (w/v) final concentration. Of the solid supports tested, TMS-250, Styrene-250, Styrene60, ODS-120A, and ODS-120T have hydrophobie surface properties; the rest of the solid supports tested were hydrophilic. Particle size diameters were 5 /zm except for TMS-250, which had a diameter of 10 /zm [181. As shown in Fig. 1, the amount of glucosylceramide produced on the surface was relatively greater when hydrophilic rather than hydrophobic gel was used, and Silica-60 gel exhibited the highest incorporation of radio-labeled glucose into the ceramide substrate. We therefore chose Silica-60 gel as the solid support for the following experiments.
Effect of ceramide density on [14C]glucose incorporation To evaluate the optimal surface concentration of ceramide, we performed the enzyme assay using ceramide adsorbed to silica gel at densities varying from 0 to 200/zg per mg of silica gel. The detergent CHAPS was again used provisionally at a concentration of 1% 100
8 o
8
c
--~ E
50
N 0
%
% %%
%%
Fig. 1, [14C]Glucose incorporation to ceramide adsorbed on various solid supports. Each solid support was coated with ceramide at a density of 20 p,g of ceramide per mg of solid. 1 mg of these solid substrates and 78 /zg of protein were used per assay. CHAPS concentration was 1% (w/v). Maximum activity corresponded to an activity of 1.54 n m o l / m g of protein per h.
0
I
2
L___
5
10
50
i
_ __
100
Ceramide density (mgceramide/gsilicagel)
Fig. 2. Effects of ceramide density on [t4C]glucose incorporation by rat brain UDP-glucose:ceramide glucosyltransferase. Silica gel was coated with various concentrations of ceramide to yield the ceramide density indicated. 1 mg of each solid substrate was used per assay. The assay conditions were the same as those outlined in Fig. 1. The actual incorporation of glucose at the maximum (100%) was 0.78
nmol/mg membraneprotein per h.
(w/v). Fig. 2 shows that [14C]glucose incorporation increased with increasing ceramide density up to 20 /zg/ml silica gel, reached a plateau, and then decreased. Therefore we fixed the ceramide density at 20 /zg/mg silica gel for the following experiments.
Effects of detergents on [14C]glucose incorporation Fig. 3 shows the effects of various detergents at varying concentrations on [14C]glucose incorporation into ceramide immobilized on a silica gel surface. Whereas the incorporation of radio-labeled glucose to ceramide was reduced by most of the detergents at any concentrations tested, only CHAPS, a zwitterionic detergent, exhibit a significant stimulatory effect at a concentration of 1% (w/v). This characteristic stimulatory and inhibitory profile was also observed with CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-l-propanesulfonate), a zwitterionic detergent similar in structure to CHAPS. When re-measured at a narrower concentration range, the CHAPS concentration achieving maximum incorporation was found to be 1% (w/v) (inset to Fig. 3). Because the reduction of glucose incorporation at higher concentrations of detergents might also result from the release or solubilization of ceramide from the solid support, an alternative assay method previously reported [5,16] was also carried out to assess the effect of detergents on the incorporation of glucose into ceramide. As shown in Table I, the stimulatory and inhibitory pattern observed in this micellar system was similar to that seen in the solid phase procedure. The effect of CHAPS concentration on the release of ceramide from the solid support was further studied using radiolabeled ceramide. [14C]Palmytoyl sphingo-
100 100
........................
•
