©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of UDP-glucose:Ceramide Glucosyltransferase from Rat Liver Golgi Membranes (*)

(Received for publication, July 10, 1995; and in revised form, October 31, 1995)

Pascal Paul Yasushi Kamisaka (§) David L. Marks Richard E. Pagano (¶)

From the Mayo Clinic and Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We present a method for solubilizing and purifying UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)) from rat liver and present data on its substrate specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with 3[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate to solubilize the enzyme. GCS activity was monitored throughout purification using UDP-Glc and a fluorescent ceramide analog as substrates. Purification of GCS was achieved via a two-step dye-agarose chromatography procedure using UDP-Glc to elute the enzyme. This resulted in an enrichment >10,000-fold relative to the starting homogenate. The enzyme was further characterized by sedimentation on a glycerol gradient, I labeling, and SDS-polyacrylamide gel electrophoresis, which demonstrated that two polypeptides (60-70 kDa) corresponded closely with GCS activity. Purified GCS was found to require exogenous phospholipids for activity, and optimal results were obtained using dioleoyl phosphatidylcholine. Studies of the substrate specificity of the purified enzyme demonstrated that it was stereospecific and dependent on the nature and chain length of the N-acyl-sphingosine or -sphinganine substrate. UDP-Glc was the preferred hexose donor, but TDP-glucose and CDP-glucose were also efficiently used. This study provides a basis for molecular characterization of this key enzyme in glycosphingolipid biosynthesis.


INTRODUCTION

Glycosphingolipids (GSLs) (^1)are amphipathic molecules that contain the hydrophobic moiety, ceramide, and a hydrophilic oligosaccharide residue. They are found in the plasma membranes of all eukaryotic cells and play important roles in cell recognition, cell proliferation and differentiation, immune recognition, and signal transduction (for reviews, see (1, 2, 3, 4, 5) ).

The biochemical pathways for GSL synthesis are well established (reviewed in (6, 7, 8, 9) ), but not all of the enzymes involved in GSL synthesis have been purified and/or cloned (for review, see (10) ). One such enzyme is UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)), which catalyzes the formation of glucosylceramide (GlcCer) from ceramide and UDP-Glc(11) . This enzyme is of particular interest for a number of reasons. First, it has been shown that there is a correlation between tumor progression and cell surface GSLs(1, 12) . Since many complex acidic and neutral GSLs are derived from GlcCer, regulation of GCS activity could have a profound effect on cell growth activity. Indeed, Radin and colleagues (13) have developed a GCS inhibitor (1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)) and demonstrated some remarkable effects of this compound both in vitro and in vivo. Recently, a defect in GCS activity was also characterized in a mutant melanoma cell line and associated with altered growth properties(14) . Second, GCS activity is concentrated at the Golgi complex(15, 16) , but its precise distribution is not known. Unlike sphingomyelin synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis is more widely distributed within the Golgi. Thus, it is of interest to learn what parts of the GCS molecule are responsible for its unique intracellular distribution. Finally, it should be noted that GlcCer synthesis occurs at the cytosolic surface of intracellular membranes(15, 16, 17) . However, formation of complex GSLs from GlcCer is believed to occur by glycosylation reactions taking place on the lumenal surface of the Golgi apparatus. Thus, GlcCer synthesis must be accompanied by transbilayer movement or ``flip-flop'' of the lipid during or after its synthesis.

To further study some of these problems, it will be necessary to purify, characterize, and eventually clone and sequence GCS. In the present paper, we describe methods for the solubilization and purification of GCS from rat liver Golgi membranes as a step toward this goal. We also provide novel information about the enzymatic properties of GCS.


EXPERIMENTAL PROCEDURES

Materials

Male Sprague-Dawley rats (5 weeks old) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). N-[7-(4-Nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D-erythro-sphingosine (C(6)-NBD-Cer) and N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C(5)-DMB-Cer) were obtained from Molecular Probes. The four steroisomers of C(6)-NBD-Cer were synthesized and purified as described(18) . Other fluorescent and radioactive ceramide analogues were prepared by N-acylation of sphingosine (or sphinganine) using N-hydoxysuccinimidyl fatty acids(18, 19) . D-threo-PDMP was from Matreya, Inc. (Pleasant Gap, PA). CHAPSO was from Pierce. Green dye-Sepharose CL-2B, hereafter referred to as ``dye-agarose,'' was prepared from active Green 19 dye (Sigma) and Sepharose CL-2B (Pharmacia Biotech Inc.) as described(20) . Phospholipids were from Avanti Polar Lipids, Inc. (Alabaster, AL). Bovine liver catalase, equine heart cytochrome C, rabbit muscle lactate dehydrogenase, and porcine heart malate dehydrogenase were obtained from Calbiochem. I was from DuPont NEN. Organic solvents were from Burdick & Jackson Laboratories Inc. (Muskegon, MI). All other chemicals and reagents were from Sigma.

Preparation of Golgi Membranes

Golgi-enriched membranes from rat liver were prepared as described (21, 22) with the following modifications. Livers were minced with a razor blade in homogenization buffer (0.25 M sucrose, 50 mM Tris/HCl, pH 7.4, 25 mM KCl containing 1 µg/ml each of antipain and leupeptin, 25 µM amidinophenylmethanesulfonyl fluoride, and 10 µg/ml aprotinin). Additional homogenization buffer was added to make a 20% (w/v) suspension, and the minced livers were then homogenized by passing the suspension through a ball bearing homogenizer four times (Berni-Tech Engineering, Saratoga, CA) with a clearance of 0.0054 inches(23) . The homogenate was adjusted to 1.07 M sucrose by addition of 2.0 M sucrose. The homogenate (19 ml) was loaded into ultracentrifuge tubes and overlaid in succession with 4 ml of 1.02 M sucrose and a 14-ml linear gradient of 1.02 M to 0.2 M sucrose. After centrifugation (Beckman SW28 rotor, 83,000 times g, 4 °C, 210 min), a prominent white band at 0.75 M sucrose was harvested. These Golgi-enriched fractions were flash frozen in liquid nitrogen and stored at -80 °C until use. Under these conditions, GCS activity was stable for several months.

Purification of UDP-Glc:Ceramide Glucosyltransferase

All purification steps were performed at 4 °C. All buffers used throughout the purification contained a mixture of protease inhibitors (1 mM EDTA, 1 µg/ml antipain, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 mM amidinophenylmethanesulfonyl fluoride, 130 mM bestatin, 1 mM pepstatin).

Detergent Solubilization of Membrane Proteins

Golgi-rich fractions from rat liver were thawed and mixed with an equal volume of 0.1% N-lauroylsarcosine in buffer A (50 mM Hepes, pH 7.4, 20% (v/v) glycerol, and 0.02% NaN(3)) with 25 mM KCl and 2 mM dithiothreitol. The mixture was stirred for 30 min and centrifuged for 1 h at 200,000 times g (Beckman 50.2Ti rotor). GCS activity was recovered in the N-lauroylsarcosine-insoluble pellet. The pellet was suspended at 1 mg of protein/ml in buffer A supplemented with 1% CHAPSO, 1 mM UDP-Glc, and 1 mM dithiothreitol and stirred for 1 h. The CHAPSO-soluble supernatant and insoluble pellet were separated after centrifugation for 1 h at 100,000 times g.

Dye-Agarose Chromatography

Preliminary studies showed that GCS bound to dye-agarose but was eluted from the dye column in the presence of UDP-Glc. These observations suggested a two-step procedure to enrich for GCS. First, CHAPSO-soluble supernatant (50 ml) was loaded (flow rate of 12 ml/h) onto a dye-agarose column (1 times 20 cm), which had been equilibrated with buffer B (buffer A supplemented with 0.5% CHAPSO and 1 mM dithiothreitol) plus 1 mM UDP-Glc. The unbound fraction (42 ml) from the first column containing 70% of the total applied GCS activity was concentrated to 4 ml by using Centriprep-50 concentrators (Amicon, Inc.). UDP-Glc was then removed from the unbound fraction by gel filtration through Biogel P2 (Bio-Rad) columns (1 times 10 cm) equilibrated with buffer B using a centrifugation method(24) . The unbound fraction was then slowly applied (12 ml/h) onto a second dye-agarose column (1 times 4 cm) equilibrated in buffer B. The column was washed with 10 ml of buffer B and sequentially eluted with 20 ml of 0.15 M KCl in buffer C (buffer B plus 0.1 mM 1,2-dioleoyl phosphatidylcholine (DOPC)), 15 ml of 20 mM UDP-Glc, and 20 mM NADH in buffer C followed by 15 ml of 1 M KCl in buffer C. 2-ml fractions were collected, and aliquots (20 µl) of each fraction were assayed for GCS activity.

Glycerol Density Gradients

Purified GCS samples were first chromatographed through a Sephadex G-25 column (1 times 5 cm) equilibrated with 1% glycerol in buffer D (50 mM Hepes, pH 7.4, 0.6% CHAPSO, 0.5 mM UDP-Glc, and 100 mM KCl) as described above for the gel filtration procedure to reduce glycerol concentration. The fractions (250 µl) were then loaded on top of 6-25% (v/v) linear glycerol gradients in 5 ml of buffer D and centrifuged in a Beckman SW 55Ti rotor at 250,000 times g for 12 h. After centrifugation, 30 fractions of 170 µl were collected starting from the top of the gradient and were assayed for enzymatic activity. The sedimentation coefficient of the native enzyme was estimated as described previously (25) using catalase, cytochrome C, malate dehydrogenase, and lactate dehydrogenase as calibrating enzymes. For polypeptide visualization, an aliquot of the enzyme fraction was prelabeled with I (see below) and added as a tracer to the sample loaded onto the glycerol gradient. The radioactive polypeptides in the glycerol gradient fractions were then analyzed by SDS-PAGE (see below).

GCS Assay

GCS activity was assayed as described previously(16) , with the following modifications. Enzyme fractions were preincubated in screw cap tubes in 50 mM Hepes (pH 7.4), 25 mM KCl, 5 mM MnCl(2), 5 mM UDP-Glc in a final volume of 0.5 ml for 5 min at 37 °C. The enzyme reaction was initiated by addition of 5 nmol of C(6)-NBD-Cerbulletbovine serum albumin complex prepared as described(18, 26) . After 15 min at 37 °C with constant stirring, the reaction was stopped by addition of 3 ml of chloroform/methanol (1:2 (v/v)). Lipids were extracted (27) and separated by thin layer chromatography(26) . Individual spots on the TLC plates were identified by comparison with fluorescent standards and quantified by image analysis as described(28) . In some cases, the reaction tubes were precoated with phospholipids (usually 0.1 mM DOPC) by first adding phospholipid dissolved in chloroform to screw cap tubes and evaporating the chloroform under a stream of N(2). For the experiment shown in Fig. 5, the reaction tubes were also precoated with D-threo-PDMP using an ethanolic stock solution of the inhibitor.


Figure 5: Effect of D-threo-PDMP on GCS activity. Dye-agarose-purified GCS was preincubated with 2.5 µmol of UDP-Glc, 0.05% CHAPSO, and 0.1 mM DOPC for 5 min at 37 °C in the presence of D-threo-PDMP. The reaction was initiated by addition of 5 nmol of C(6)-NBD-Cer, and the results were quantitated as described in Table 2and under ``Experimental Procedures.'' The data are means of triplicate determinations and are expressed as a percent of control values.





Analytical Methods

The polypeptide composition of various fractions was assessed by SDS-PAGE(29) . In some cases, the enzyme fractions were concentrated by precipitation with trichloroacetic acid in the presence of sodium deoxycholate prior to electrophoresis(30) . Polypeptides were visualized using a silver staining kit (Bio-Rad) as described(31) . To visualize the polypeptides in samples with very low protein concentrations, samples were prelabeled with 0.5-1 mCi of I by the chloramine-T method(32) . Radioactive gels were analyzed using a phosphorimager (Molecular Analyst GS-363, Bio-Rad) and by autoradiography. Immunoprecipitation of rat albumin was performed as described (33) using a polyclonal anti-rat albumin antiserum from Cappell/Organon Teknika (Durham, NC). The Golgi marker enzyme, galactosyltransferase, was measured as described(35) . Electron microscopy of Golgi membranes was performed as described(21) . Protein concentrations were measured using Coomassie Blue dye reagent (Bio-Rad) with bovine serum albumin as a standard(36) .


RESULTS

Purification of GCS

Preliminary studies showed that GCS in Golgi membranes could be solubilized with CHAPSO (1% (w/v)) with recovery of 70% of the activity in the extract (data not shown). We also found that addition of 0.1-1.0 mM DOPC and 0.1 mM UDP-Glc to the solubilized GCS improved its stability at 4 °C (data not shown). Thus, these protective agents were added during some purification steps. Finally, we noted that after chromatographic procedures, GCS had specific phospholipid requirements for optimal activity (see below). Thus, most activity assays of solubilized GCS fractions were performed in the presence of 0.1 mM DOPC.

We used rat liver Golgi membranes as a starting material for the purification of GCS because our previous studies showed that these membranes were highly enriched in GCS activity(16) . The Golgi fractions prepared in the present work were enriched 60-80-fold in galactosyltransferase, a trans-Golgi marker protein, with 35% recovery of this enzyme. In addition, electron microscopy of the Golgi samples revealed numerous intact stacks comprised of 4 and 5 cisternae (data not shown). Thus, these Golgi preparations were similar in enrichment and morphology to highly purified Golgi fractions prepared by established methods (for review, see (37) ).

Golgi membrane fractions were enriched >60-fold in GCS activity compared to crude rat liver homogenate with a yield of >50% (Table 1). Since our enzyme assay used a fluorescent ceramide analog, which is also a substrate for sphingomyelin synthase(18, 38, 39) , we were also able to determine that the Golgi membranes were enriched 35-40-fold in sphingomyelin synthase activity with a recovery of 40% (data not shown). This observation is in agreement with the differential location of GCS and sphingomyelin synthase in the Golgi apparatus(16, 17, 21, 40) .



Detergent Solubilization

We found that pretreatment of Golgi-enriched fractions with 0.05% (w/v) N-lauroylsarcosine followed by ultracentrifugation resulted in the removal of 75% of the total Golgi protein in the supernatant, while most GCS activity (80%) was recovered in the pellet (Table 1). The enrichment achieved by this step was 200-fold relative to homogenate (Table 1) or 3.15-fold compared to Golgi membranes. GCS activity in the pellet was then solubilized with 1% CHAPSO. The solubilized GCS was not apparently further enriched (Table 1). However, activity of the solubilized enzyme was tested in the presence of CHAPSO (final concentration, 0.05%), a condition that suppresses GCS activity in intact Golgi membranes by 30% (data not shown). Thus, the % recovery and enrichment values shown in Table 1are underestimates.

Two-step Dye-Agarose Column Chromatography

We attempted to purify GCS by affinity chromatography using various bound ligands (UDP-hexanolamine, UDP-GlucUA, ceramide, sphingosine, and PDMP), but these efforts were unsuccessful. We next explored dye adsorption chromatography and found that GCS activity could be released from a dye-agarose column by UDP-Glc, while most other dye-binding proteins were elutable only with salt. Thus, we devised a two-step purification procedure. First, CHAPSO-solubilized Golgi membranes were loaded onto the dye-agarose column in the presence of 1 mM UDP-Glc. GCS activity was then recovered (70% of total applied activity) in the unbound fractions with a 2.3-fold enrichment relative to the CHAPSO extract or a 474-fold relative to the homogenate (Table 1; Fig. 1A). After gel filtration to remove UDP-Glc, the unbound fractions from the first dye-agarose column were loaded onto a second dye-agarose column, which was equilibrated with buffer without UDP-Glc. Although most proteins passed through the second column, almost all the GCS activity was bound and was eluted successively with 0.15 M KCl, 20 mM UDP-Glc plus 20 mM NADH, and 1 M KCl (Fig. 1B). The 0.15 M KCl-eluted fraction was enriched 5000-fold in GCS activity with a 1.15% recovery relative to homogenate (Table 1). The amount of protein recovered in the UDP-Glc-eluted fraction was at the lower limits of detectibility (1 µg/ml), resulting in a geq10,000-fold enrichment of GCS with a recovery of leq0.5% (Table 1). The 1 M KCl-eluted fraction was also significantly enriched (3300-fold) in GCS activity (Table 1). Furthermore, since CHAPSO partially inhibits GCS activity as noted above, the true enrichment of GCS in the purified fractions is greater than the values cited above (see Table 1).


Figure 1: Chromatography of rat liver Golgi GCS on dye-agarose. The detergent-solubilized enzyme was loaded onto a dye-agarose column (A) in the presence of 1 mM UDP-Glc as described under ``Experimental Procedures.'' After washing with 50 ml of buffer B and 50 ml of 0.15 M KCl in buffer B without UDP-Glc, the column was eluted with 1 M KCl in the same buffer as indicated by the arrow. The unbound fractions, indicated by the horizontal bar, were pooled, subjected to gel filtration, and loaded onto a second dye-agarose column equilibrated in buffer B without UDP-Glc (B). The column was then washed in the same buffer and sequentially eluted (at arrows) with 0.15 M KCl (1), 20 mM UDP-Glc plus 20 mM NADH (2), and 1 M KCl (3) in buffer C.



Polypeptide Composition of GCS Fractions

The polypeptide composition of GCS fractions during different steps of purification was assessed by SDS-PAGE (Fig. 2). Initial steps (Fig. 2, lanes 1-4) were visualized by silver staining. To visualize the fractions eluted from the second dye-agarose column, which were very low in protein, aliquots were radioiodinated. When a radiolabeled aliquot of the 0.15 M KCl-eluted fraction was electrophoresed and visualized by autoradiography, the profile obtained (Fig. 2, lane 5) showed a similar pattern to that of the same sample visualized by silver staining in lane 4. Three major bands (45, 60, and 66 kDa) were visible in this fraction (Fig. 2, lane 5). The 45-kDa polypeptide was noticeably diminished in the UDP-Glc-eluted fraction, while the 60- and 66-kDa forms continued to predominate on the gel (Fig. 2, lane 6).


Figure 2: SDS-PAGE analysis of GCS at various steps of purification. Protein samples from different steps in the purification of GCS from rat liver Golgi membranes were subjected to SDS-PAGE under reducing conditions using a 10% gel. Following electrophoresis, the gel was either silver stained (lanes 1-4) or I-labeled proteins were visualized by autoradiography (lanes 5 and 6). Lane 1, CHAPSO extract; lane 2, unbound fraction from dye-agarose (column I); lane 3, unbound fraction from dye-agarose (column II); lanes 4 and 5, 0.15 M KCl elution from dye-agarose (column II); lane 6, UDP-Glc/NADH elution from dye-agarose (column II). Approximately 5 µg of protein was loaded in each of lanes 1-3 and 1.5 µg in lane 4. Arrows indicate the positions of molecular weight standards.



In an attempt to further purify GCS following dye-agarose chromatography, UDP-Glc-eluted GCS, including a radiolabeled aliquot, was centrifuged through a glycerol gradient. Preliminary experiments showed that only 50% of the starting GCS activity was preserved after a 12-h ultracentrifugation in a glycerol gradient with 0.5 mM UDP-Glc present, preventing a quantitative evaluation of GCS enrichment. Nevertheless, the discrete peak of GCS activity in the glycerol gradient allowed us to assess the apparent relative size of the enzyme under nondenaturing conditions. Based on a standard curve of marker enzymes sedimented in the same tube, we estimated the sedimentation coefficient of GCS as 4.2 s (Fig. 3A). The peak of GCS activity migrated to position immediately preceding malate dehydrogenase, which has a molecular mass of 70 kDa. Fig. 3B shows the glycerol gradient distribution of the radiolabeled 45-, 60-, and 66-kDa polypeptides present in dye-agarose purified GCS. The intensity of the 45-kDa polypeptide peaked earlier in the glycerol gradient than the peak of GCS activity (Fig. 3, B and C). Most of the radioactivity associated with the 66-kDa band was found to be rat albumin by immunoprecipitation; however, this technique did not completely remove the radioactivity in this region of the gel (data not shown). Furthermore, both the 60- and the 66-kDa bands peaked in intensity at fraction 11, which coincided with the peak of GCS activity (Fig. 3, B and C). Thus, although we cannot definitively rule out the possibility that another polypeptide is responsible for GCS activity, our data suggest that the 60- and/or 66-kDa polypeptides are the GCS protein.


Figure 3: Glycerol gradient sedimentation of purified GCS. A concentrated sample of dye-agarose-purified GCS (250 µl) including an aliquot (4 times 10^6 cpm), which had been radiolabeled with I, was layered onto the top of a 6-25% glycerol density gradient (5 ml) and centrifuged at 250,000 times g for 12 h as described under ``Experimental Procedures.'' The gradient was then fractionated into 30 samples, and the fractions were assessed for polypeptide composition, GCS activity, and the positions of marker proteins (cytochrome C, 14 kDa; malate dehydrogenase (MDH), 70 kDa; lactate dehydrogenase (LDH), 140 kDa; and catalase, 250 kDa) as described under ``Experimental Procedures.'' A, standard curve showing the migration of marker proteins and GCS activity. B, 10% SDS-PAGE of glycerol gradient fractions in which I-labeled proteins were visualized by a phosphorimager. AS, applied sample. Numbers beneath the gel correspond to glycerol gradient fractions. Position of molecular mass markers are shown at the right. Arrow at left marks position of the 60-kDa polypeptide. C, relative GCS activity plotted versus glycerol gradient fraction number. Also shown plotted versus fraction number are the relative intensities of the 45-, 60-, and 66-kDa polypeptides determined by phosphorimager analysis.



Characterization of GCS

Purified fractions obtained after the second dye-agarose column were used to examine the enzymatic characteristics and substrate specificity of GCS. GCS activity was previously reported to be stimulated by phospholipids(41, 42) . Thus, we first investigated effects of phospholipids on GCS activity. Purified GCS showed an almost absolute requirement for phospholipids for activity (Table 2, Fig. 4). In contrast, GCS activity in the Golgi and crude CHAPSO extract was not stimulated significantly by phospholipids (data not shown). The optimal phospholipid concentration for stimulating GCS activity in purified fractions was 0.1 mM, with higher concentrations causing a relative suppression of activity (Fig. 4). Of the phospholipids tested, phosphatidylcholines showed the greatest ability to stimulate GCS activity, although phosphatidylethanolamine was almost as effective (Table 2). Among phosphatidylcholines with different fatty acid moieties, GCS activity was maximally stimulated by those containing unsaturated fatty acids, with C and C fatty acids being the most effective (Table 2).


Figure 4: Effect of phospholipid concentration on GCS activity. Dye-agarose-purified GCS, eluted in the absence of exogenous phospholipids, was incubated in the presence of various concentrations of synthetic (di-C, di-C and C, C) phosphatidylcholines as described in Table 2.



Next we examined the specificity of GCS for various acceptor substrates. Among the ceramide analogs tested, the D-erythro- and L-erythro- isomers of C(6)-NBD-Cer were the best substrates (Table 3). Glucosylation of C(6)-NBD-Cer from a commercial source, which contains a mixture of both isomers, gave similar results to the pure stereoisomers (Table 3). Neither D- nor L-threo-NBD-Cer were used as substrates by the enzyme, showing that the enzyme is stereospecific. D-erythro-C(6)-NBD-dihydroceramide was glucosylated to about 25% of control values obtained with C(6)-NBD-Cer (Table 3). Among the different C(n)-NBD-Cer tested, the hexanoyl- and octanoyl- analogs were the best GCS substrates, while little or no measurable activity was found toward shorter (propanoyl- and pentanoyl-) and longer (tetradecanoyl-) ceramide analogs (Table 3). Similarly, GCS was 4.5 times more active toward short chain [^14C]C(6)-Cer than toward a longer chain [^14C]C-Cer (data not shown). (^2)However, the glucosylation yield of the radioactive C(6)-Cer was only 25% of that of C(6)-NBD-Cer (data not shown). By contrast, C(5)-DMB-Cer containing a different fluorophore was a poor substrate with 21% glucosylation compared to C(6)-NBD-Cer (Table 3). We also tested the effects of D-threo-PDMP, a known specific inhibitor of GCS (43) . PDMP inhibited the activity of purified GCS 45% at 1 µM and 85% at 10 µM (Fig. 5).



Finally, we evaluated the ability of GCS to use various donor substrates to glycosylate C(6)-NBD-Cer. Of the UDP-hexoses, GCS was able to utilize UDP-Glc efficiently but had little or no activity using UDP-glucuronic acid, UDP-galactose, UDP-N-acetylglucosamine, UDP-mannose, or UDP-xylose as hexose donors (Table 4). UDP-Glc was the best glucose donor among diphosphoglucose nucleotides, but TDP-glucose and CDP-glucose also were efficient glucose donors (Table 4). Surprisingly, ADP-glucose was also used as a substrate by GCS, leading to about 6% glucosylation of C(6)-NBD-Cer compared to UDP-Glc.




DISCUSSION

We report here for the first time a method for the purification of GCS. Several features of the method were critical for the successful purification of this enzyme. First, the modifications that we introduced in the homogenization and Golgi fractionation procedure (see ``Experimental Procedures'') significantly improved the enrichment and recovery of GCS activity relative to our previous work(16) . Second, we found that inclusion of UDP-Glc and DOPC as protective agents improved the stability of GCS. Similarly, UDP and phospholipids were reported to stabilize UDP-Glc:dolichyl-phosphate glucosyltransferase(44) ; however, UDP had no protective effect on GCS activity (data not shown). Finally, we found that green dye-agarose could be used in a two-step procedure to purify GCS based on the selective binding of GCS to the dye-agarose in the absence, but not in the presence, of UDP-Glc. This procedure led to a 20-fold enrichment of GCS in the UDP-Glc-eluted fractions and was the only chromatographic procedure that we tried that produced any enrichment in the enzyme. Dye-agaroses have been used previously in the purification of numerous proteins such as dehydrogenases, kinases, and serum proteins (for review, see (45) ) as well as UDP-Glc:dolichyl-phosphate glucosyltransferase(46) . The observation that the binding of GCS to dye-agarose is inhibited by UDP-Glc suggests a competition between UDP-Glc and dye molecules for the active site of the enzyme. Similar results were described for another glycosyltransferase purified on dye-agarose(47) .

Using the solubilized and purified GCS, we also obtained new information about the enzymatic characteristics of this protein. First, we found that purified GCS had almost no activity in the absence of exogenous phospholipid. Activity was restored by the addition of phospholipids, with the highest enhancement of activity observed with low concentrations (0.1 mM) of unsaturated, long chain (C or C) phosphatidylcholine (Table 2, Fig. 4). In contrast, phospholipids had little effect on GCS activity in the Golgi or CHAPSO-solubilized Golgi membranes. Presumably, endogenous phospholipids present in Golgi membranes were sufficient to stimulate maximal activity in the Golgi fractions. These endogenous phospholipids may have been depleted during purification of the enzyme by dye-agarose chromatography, causing an almost complete loss of GCS activity, which could be restored by exogenous phospholipids. A stimulating effect of exogenous phospholipids on glycosyltransferase activities was documented previously(42, 48, 49, 50) . More recently, stimulation of solubilized GCS activity by phosphatidylcholine was reported(51) ; however, the concentrations reported for optimal activity were 100-fold higher (8-10 mM) than the value that we report here (0.1 mM).

Our results on the specificity of purified GCS toward ceramide analogs are in good agreement with previously published studies concerned with GCS activity in cultured cells or membrane preparations. First, we found that GCS is stereospecific, utilizing erythro- but not threo-C(6)-NBD-Cer as a substrate, similar to results reported using cultured fibroblasts(18) . We also found that D-erythro-C(6)-NBD-dihydroceramide was glucosylated to about 25% of control values obtained with C(6)-NBD-Cer (Table 3). This result supports recent observations on the metabolism of C(6)-Cer analogs in Chinese hamster ovary cells(52) . Second, we found that C(6)- and C(8)-ceramides were better substrates than ceramides with longer or shorter N-acyl chains, confirming observations reported for GCS activity in microsomal preparations(41) . Finally, we found that C(6)-NBD-Cer is a better substrate for GCS than is [^14C]C(6)-Cer. By contrast, C(5)-DMB-Cer, containing a different fluorophore, was poorly glucosylated relative to C(6)-NBD-Cer (Table 3), consistent with previous findings in cultured cells(53) . We also examined the nucleotide specificity of GCS and found that, surprisingly, GCS is able to efficiently utilize CDP-Glc and TDP-Glc as glucose donors (see Table 4). While CDP-Glc and TDP-Glc are not naturally occurring glucose donors, earlier studies have also described UDP-Glc:glucosyltransferases that are able to utilize CDP-Glc and TDP-Glc(54, 55) .

In summary, we have presented for the first time a method for the purification of GCS, applied this method to isolate a 10,000-fold enriched GCS fraction from rat liver, identified two polypeptides (60-70 kDa) as likely candidates for the GCS protein, and further characterized the purified enzyme. This information provides a basis for future molecular studies of this key enzyme in GSL biosynthesis.


FOOTNOTES

*
This work was supported by U. S. Public Health Service Grant R37 GM22942. Part of this work was carried out at the Carnegie Institution of Washington, Dept. of Embryology (Baltimore, MD). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Applied Microbiology, National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba-shi, Ibaraki 305, Japan.

To whom correspondence should be addressed: Mayo Clinic and Foundation, Guggenheim 6, 200 First St., SW, Rochester, MN 55905-0001. Tel.: 507-284-8754; Fax: 507-284-4521; :pagano.richard{at}mayo.edu.

(^1)
The abbreviations used are: GSL, glycosphingolipid; Cer, ceramide; CHAPSO, 3[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate; C(5)-DMB-Cer, N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-sphingosine; C(6)-NBD-Cer, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-D-erythro-sphingosine; DOPC, 1,2-dioleoyl phosphatidylcholine; GCS, glucosylceramide synthase; GlcCer, glucosylceramide; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PAGE, polyacrylamide gel electrophoresis.

(^2)
We were unable to test the activity of purified GCS using long-chain, naturally occurring ceramides because of their insolubility in our assay solution.


ACKNOWLEDGEMENTS

-We thank the members of the Pagano laboratory for encouragement and critical reading of the manuscript.

Note Added in Proof-Recently Ichikawa et al. (34) have isolated a cDNA encoding human GCS by expression cloning using GM-95 cells lacking the enzyme as a recipient. The open reading frame encodes a protein containing 394 amino acids (predicted molecular mass of 44.9 kDa).


REFERENCES

  1. Hakomori, S.-I. (1984) Annu. Rev. Immunol. 2, 103-126 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hakomori, S.-I. (1990) J. Biol. Chem. 265, 18713-18716 [Abstract/Free Full Text]
  3. Hannun, Y. A., and Bell, R. M. (1989) Science 243, 500-507 [Medline] [Order article via Infotrieve]
  4. Ballou, L. R. (1992) Immunol. Today 13, 339-341 [Medline] [Order article via Infotrieve]
  5. Schnaar, R. L. (1991) Glycobiology 1, 477-485 [Medline] [Order article via Infotrieve]
  6. Sweeley, C. C. (1991) in Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, D. E., and Vance, J., eds) pp. 327-361, Elsevier Press, New York
  7. van Echten, G., and Sandhoff, K. (1993) J. Biol. Chem. 268, 5341-5344 [Free Full Text]
  8. Kishimoto, Y. (1983) in The Enzymes (Boyer, P. D., ed) 3rd Ed., pp. 357-445, Academic Press, New York
  9. Kanfer, J. N. (1983) in Handbook of Lipid Research. (Kanfer, J. N., and Hakomori, S., eds) Vol. 3, pp. 167-247, Plenum Press, New York
  10. Kleene, R., and Berger, E. G. (1993) Biochim. Biophys. Acta 1154, 283-325 [Medline] [Order article via Infotrieve]
  11. Basu, S., Kaufman, B., and Roseman, S. (1968) J. Biol. Chem. 243, 5802-5807 [Abstract/Free Full Text]
  12. Hakomori, S. (1994) in Progress in Brain Research (Svennerholm, L., Asbury, A. K., Reisfeld, R. A., Sandhoff, K., Suzuki, K., Tettamanti, G., and Toffano, G., eds) Vol. 101, pp. 241-250, Elsevier, Cambridge, UK
  13. Radin, N. S., Shayman, J. A., and Inokuchi, J. (1993) in Advances in Lipid Research (Bell, R. M., Hannun, Y. A., and Merrill, A. H., Jr., eds) Vol. 26, pp. 183-213, Academic Press, New York
  14. Ichikawa, S., Nakajo, N., Sakiyama, H., and Hirabayashi, Y. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703-2707 [Abstract]
  15. Coste, H., Martel, M.-B., and Got, R. (1986) Biochim. Biophys. Acta 858, 6-12 [Medline] [Order article via Infotrieve]
  16. Futerman, A. H., and Pagano, R. E. (1991) Biochem. J. 280, 295-302 [Medline] [Order article via Infotrieve]
  17. Jeckel, D., Karrenbauer, A., Burger, K. N. J., van Meer, G., and Wieland, F. (1992) J. Cell Biol. 117, 259-267 [Abstract]
  18. Pagano, R. E., and Martin, O. C. (1988) Biochemistry 27, 4439-4445 [Medline] [Order article via Infotrieve]
  19. Schwarzmann, G., and Sandhoff, K. (1987) Methods Enzymol. 138, 319-341 [Medline] [Order article via Infotrieve]
  20. Stellwagen, E. (1990) Methods Enzymol. 182, 343-357 [Medline] [Order article via Infotrieve]
  21. Futerman, A. H., Stieger, B., Hubbard, A. L., and Pagano, R. E. (1990) J. Biol. Chem. 265, 8650-8657 [Abstract/Free Full Text]
  22. Bergeron, J. J. M., Rachubinski, R. A., Sikstrom, R. A., Posner, B. I., and Paiement, J. (1982) J. Cell Biol. 92, 139-146 [Abstract]
  23. Balch, W. E., and Rothman, R. E. (1985) Arch. Biochem. Biophys. 240, 413-425 [Medline] [Order article via Infotrieve]
  24. Fry, D. W., White, J. C., and Goldman, I. D. (1978) Anal. Biochem. 90, 809-815 [Medline] [Order article via Infotrieve]
  25. Haga, T., Haga, K., and Gilman, A. G. (1977) J. Biol. Chem. 252, 5776-5782 [Medline] [Order article via Infotrieve]
  26. Futerman, A. H., and Pagano, R. E. (1992) Methods Enzymol. 209, 437-446 [Medline] [Order article via Infotrieve]
  27. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  28. Koval, M., and Pagano, R. E. (1989) J. Cell Biol. 108, 2169-2181 [Abstract]
  29. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  30. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250 [Medline] [Order article via Infotrieve]
  31. Merrill, A. H., Jr., and Stevens, V. L. (1989) Biochim. Biophys. Acta 1010, 131-139 [Medline] [Order article via Infotrieve]
  32. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963) Biochem. J. 89, 114-123
  33. Larkin, J. M., and Palade, G. E. (1991) J. Cell Sci. 98, 205-216 [Abstract]
  34. Ichikawa, S., Sakiyama, H., Suzuki, G., Kazuya, I.-P., Hidari, J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. , in press
  35. Bretz, R., and Staübli, W. (1977) Eur. J. Biochem. 77, 181-192 [Medline] [Order article via Infotrieve]
  36. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  37. Andersson, G., and Glaumann, H. (1987) in Lysosomes: Their Role in Protein Breakdown (Glaumann, H., and Ballard, F. J., eds) pp. 61-113, Academic Press, New York
  38. Lipsky, N. G., and Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2608-2612 [Abstract]
  39. Lipsky, N. G., and Pagano, R. E. (1985) J. Cell Biol. 100, 27-34 [Abstract]
  40. Jeckel, D., Karrenbauer, A., Birk, R., Schmidt, R. R., and Wieland, F. (1990) FEBS Lett. 261, 155-157 [CrossRef][Medline] [Order article via Infotrieve]
  41. Vunnam, R. R., and Radin, N. S. (1979) Biochim. Biophys. Acta 573, 73-82 [Medline] [Order article via Infotrieve]
  42. Cestelli, A., White, F. V., and Costantino-Ceccarini, E. (1979) Biochim. Biophys. Acta 572, 283-292 [Medline] [Order article via Infotrieve]
  43. Inokuchi, J.-I., and Radin, N. S. (1987) J. Lipid Res. 28, 565-571 [Abstract]
  44. Matern, H., Bolz, R., and Matern, S. (1990) Eur. J. Biochem. 190, 99-105 [Abstract]
  45. Lowe, C. R., Small, D. A. P., and Atkinson, A. (1981) Int. J. Biochem. 13, 33-40 [Medline] [Order article via Infotrieve]
  46. Gold, P., and Green, M. (1983) J. Biol. Chem. 258, 12967-12975 [Abstract/Free Full Text]
  47. Bar-Peled, M., Lewinsohn, E., Fluhr, R., and Gressel, J. (1991) J. Biol. Chem. 266, 20953-20959 [Abstract/Free Full Text]
  48. Costantino-Ceccarini, E., and Suzuki, K. (1975) Arch. Biochem. Biophys. 167, 646-654 [Medline] [Order article via Infotrieve]
  49. Holmes, E. H., and Hakomori, S. (1987) J. Biochem. (Tokyo) 101, 1095-1105
  50. Neskovic, N. M., Sarlieve, L. L., and Mandel, P. (1974) Biochim. Biophys. Acta 334, 301-315
  51. Durieux, I., Martel, M. B., and Got, R. (1990) Int. J. Biochem. 22, 709-715 [Medline] [Order article via Infotrieve]
  52. Ridgway, N. D., and Merriam, D. L. (1995) Biochim. Biophys. Acta 1256, 57-70 [Medline] [Order article via Infotrieve]
  53. Pagano, R. E., Martin, O. C., Kang, H. C., and Haugland, R. P. (1991) J. Cell Biol. 113, 1267-1279 [Abstract]
  54. Schmid, G., and Griesbach, H. (1982) Eur. J. Biochem. 123, 363-370 [Abstract]
  55. Wojciechowski, Z. A., Zimowski, J., Zimowski, J. G., and Lyznik, A. (1979) Biochim. Biophys. Acta 570, 363-370 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.