- Email: [email protected]
Purification of Free Sphingoid Bases by Solid-Phase Extraction on Weak Cation Exchanger Cartridges
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native compared to denaturing lysis conditions were applied (Fig. 1). This was confirmed by a more detailed analysis of the time course of tyrosine phosphorylation after addition of CVI or 10% FCS (Fig. 2). Our results clearly show that if a native lysis buffer is used for immunoprecipitation of phosphotyrosinecontaining proteins, the use of a broad spectrum protein kinase inhibitor like genistein is essential to conserve the phosphorylation status of the cell at the time of lysis. In addition, the same lysate could be used for parallel detection of activated Erk2 and phosphotyrosinylated target proteins (Fig. 2). Tyrosine phosphorylation of intracellular proteins induced by diverse extracellular agents is a transient signal, often in the range of only a few minutes. Therefore, during the procedure of protein extraction, three aspects are essential: First, cell lysis must be rapid and quantitative to accurately reveal the signal intensities at different time points. For this purpose, we use liquid nitrogen to freeze the cells immediately before addition of lysis buffer, stopping all intracellular enzymatic reactions as rapidly as possible. As to the lysis conditions, these should be as gentle as possible to retain the antibody binding sites and prevent unspecific background signals, while permitting sufficient solubilization of the protein(s) of interest (10). Both criteria are met by the modified native lysis buffer. Second, the phosphorylation signal at the time of lysis must be “frozen” throughout the immunoprecipitation procedure. This is best accomplished by using a broad spectrum protein tyrosine kinase inhibitor like genistein in addition to inhibitors of proteases and phosphatases. Finally, the affinity and specificity of the anti-phosphotyrosine antibody used for immunoprecipitation is vital to yield optimal results. As established by other groups, RC20 is an antibody of choice for both immunoprecipitation and immunoblotting of phosphotyrosine-containing proteins. Acknowledgments. This project was funded by SFB 366 C5, DFG Schu 646/10-2. D. Schuppan was recipient of a Hermann-und-LillySchilling professorship. We thank Monika Schmid for expert technical assistance.
REFERENCES 1. Atkinson, J. C., Ru¨hl, M., Becker, J., Ackermann, R., and Schuppan, D. (1996) Exp. Cell Res. 228, 283–291. 2. Ru¨hl, M., Sahin, R., Johannsen, M., Somasundaram, R., Manski, D., Riecken, E. O., and Schuppan, D. (1999) J. Biol. Chem. 274, 34361–34368. 3. Ru¨hl, M., Johannsen, M., Atkinson, J., Sahin, E., Somasundaram, R., Manski, D., Riecken, E. O., and Schuppan, D. (1999) Exp. Cell Res. 250, 548 –557. 4. Ignatoski, K. M. W., and Verderame, M. F. (1996) BioTechniques 20, 794 –796. 5. Hardie, D. G. (1995) A Practical Approach (Hardie, D. G., Ed), IRL Press, Oxford.
6. Clark, E. A., and Hynes, R. O. (1996) J. Biol. Chem. 271, 14814 – 14818. 7. Huang, J., Mohammadi, M., Rodrigues, G. A., and Schlessinger, J. (1995) J. Biol. Chem. 270, 5065–5072. 8. Nahas, N., Molski, T. F., Fernandez, G. A., and Sha’afi, R. I. (1996) Biochem. J. 318, 247–253. 9. van Dijk, M. C., Hilkmann, H., and van Blitterswijk, W. J. (1997) Biochem. J. 325, 303–307. 10. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Purification of Free Sphingoid Bases by Solid-Phase Extraction on Weak Cation Exchanger Cartridges Jacques Bodennec,* ,1 Ce´cile Famy,* Ge´rard Brichon,† Georges Zwingelstein,† and Jacques Portoukalian* *Laboratory of Tumor Glycobiology, Faculty of Medicine Lyon-Sud, 69921 Oullins Cedex, France; and †Institute Michel Pacha, University Claude Bernard-Lyon I, 83500 La Seyne Sur Mer, France Received October 21, 1999
Free sphingoid bases such as sphingosine, dihydrosphingosine (sphinganine), and phytosphingosine (4-hydroxysphinganine) are extracted from cells and tissues in mixtures of solvents such as chloroform– methanol (1) and further purified by phase partition under alkaline conditions to be recovered in the organic phase (1, 2). Free sphingoid bases have been purified from neutral lipids by chromatography on small silica gel columns before further separation by HPLC (3, 4). However, by using such preparative techniques, free sphingoid bases are mostly eluted with neutral glycosphingolipids. Moreover it is often difficult to separate these molecules from each other by TLC. Solid-phase extraction (SPE) 2 could be an interesting alternative to separate free sphingoid bases from neutral glycosphingolipids. SPE is a rapid method and each recovered fraction can be directly used for further analysis, bypassing TLC separation. Here we report the use of weak cation SPE cartridges for the separation of free sphingoid bases from neutral glycosphingolipids. The SPE minicolumns are LC-WCX from Supelco (Saint Quentin Fallavier, France) which are 1 To whom correspondence should be addressed. Fax: (33) 478 86 31 48. E-mail: [email protected]. 2 Abbreviations used: SPE, solid-phase extraction; CMH, ceramide monohexosides; CDH, ceramide dihexosides; CTH, ceramide trihexosides; Globo, globosides; LCBs, long chain bases; LC-WCX, weak cation SPE cartridges.
Analytical Biochemistry 279, 245–248 (2000) doi:10.1006/abio.2000.4454 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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cosphingolipid standards were purified from human melanoma tumors according to the procedure of Saı¨to and Hakomori (5) and further fractionated by HPLC to obtain ceramide monohexosides (CMH), ceramide dihexosides (CDH), ceramide trihexosides (CTH), and globosides (Globo) (6). The SPE LC-WCX columns were preconditioned by passing successively 1 ml of hexane, 2 ml of 0.5 N acetic acid in methanol, 4 ml of methanol, and 4 ml of hexane. The lipid mixture (containing 40 g of each sphingoid base and 40 g of each neutral glycosphingolipid) was resuspended in 200 l of chloroform and applied onto the SPE tube placed on a Visiprep vacuum apparatus (Supelco). Pressure was not used before all the applying solvent was fully passed throughout the column. Then, the neutral glycosphingolipids were recovered in the first tube by eluting the column with 3 ml of chloroform:methanol (9:3, v/v) at a flow rate of 0.3 ml/min (see Fig. 1a). With such eluting solvent, the free long chain bases (LCBs) remain on the column (see Fig. 1b). The LCBs were eluted in a second fraction by washing the column with 2 ml of 1 N acetic acid in methanol. The two collected fractions were dried off under nitro-
FIG. 1. Thin-layer chromatography of the different fractions eluted from the preconditioned (see text) 100 mg LC-WCX cartridge. A mixture of neutral glycosphingolipids and free sphingoid bases is loaded on the column. The first fraction (fraction 1) is obtained by eluting the column with 2 ml chloroform:methanol (9:3, v/v). The second fraction (fraction 2) is obtained after elution of the column with 2 ml of 1 N acetic acid in methanol. The two fractions are dried under nitrogen and taken up in a small amount of chloroform: methanol (2:1, v/v). Each fraction is applied by half on two different HPTLC plates. One plate is developed in chloroform:methanol:water (65:25:4, v/v/v) and sprayed with orcinol reagent (a), and the second plate is developed in chloroform:methanol:2 N ammonia (40:10:1, v/v/v) and then visualized with ninhydrin reagent. Std in a: standard mixture of neutral glycosphingolipids CMH, CDH, CTH, and Globo. Std in b: standard mixture of free sphingoid bases: sphingosine, dihydrosphingosine, phytosphingosine. Lane 1, fraction 1; Lane 2, fraction 2.
made of a silica gel matrix (100 mg) bonded with propanoic acid in a sodium salt form. Such type of bonded phase was selected because, under appropriate conditions, the carboxylic acid functional groups on the column should bind to the amino group of free sphingoid bases with a greater relative affinity than to neutral glycosphingolipids, thus allowing separate elution of these compounds. Sphingoid base standards (D-sphingosine, DL-dihydrosphingosine, and phytosphingosine) were from Sigma (Saint Quentin Fallavier, France). Neutral gly-
FIG. 2. HPTLC plate of long-chain bases extracted and purified on LC-WCX cartridge after acid hydrolysis of both free ceramides and neutral glycosphingolipids from fresh human melanoma tumors separately isolated by aminopropyl column chromatography and TLC according to previously published procedures (9, 10). The ceramide spots and neutral glycosphingolipids are scrapped from the plate and hydrolyzed at 70°C in the presence of 1 ml of methanol:water:conc HCl (100:9.4:8.6, v/v/v) during 18 h. After alkalinization to pH 12 with 2 N KOH, the mixture is extracted with 6 ml of chloroform: methanol (2:1, v/v). The chloroformic lower phase is recovered and passed through a preconditioned LC-WCX cartridge (see text). Sphingoid bases are washed and extracted from the LC-WCX column according to the procedure described in this report and an aliquot of fraction 2 is run on HPTLC plate in chloroform:methanol:2 N ammonia (40:10:1, v/v/v). The plate is visualized with ninhydrin reagent. Lane 1, sphingoid bases from hydrolyzed free ceramides isolated from fresh human melanoma tumors; Lane 2, phytosphingosine standard; Lane 3, dihydrosphingosine standard; Lane 4, sphingosine standard; Lane 5, sphingoid bases from hydrolyzed neutral glycosphingolipids purified from fresh human melanoma tumors. The asterisk (*) shows O-methyl derivatives of LCBs.
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gen and taken up in 100 l chloroform:methanol (2:1, v/v) which were separately applied by half on two different HPTLC plates (silica gel 60 from Merck, Darmstadt, Germany) to resolve free LCBs on the one hand and neutral glycosphingolipids on the other hand. The solvent system was chloroform:methanol:2 N ammonia (40:10:1, v/v/v) for LCBs (7) and chloroform:methanol: water (65:25:4, v/v/v) for neutral glycosphingolipids. LCBs on the HPTLC plate were visualized with ninhydrin reagent and neutral glycosphingolipids with orcinol reagent. Figure 1a shows that the neutral glycosphingolipids were fully recovered in the first fraction. No glycosphingolipid could be detected in the second fraction by spraying the HPTLC plate with orcinol reagent, indicating a complete recovery of these lipids in fraction 1. In a same way, it can be seen on Fig. 1b that free sphingoid bases are recovered in the second fraction since the HPTLC plate of the first fraction is clearly ninhydrin-negative. To ensure strong binding, the use of 0.5 N acetic acid in methanol during the preconditioning procedure is required to activate the propanoic acid groups and to remove sodium ions which are present on the column matrix. Resolution of neutral glycosphingolipids from free sphingoid bases was also possible by using acetone: methanol (9:2.4, v/v) instead of chloroform:methanol. However, the solvent mixture containing chloroform was more adequate since free phytosphingosine was sometimes degraded on the column when using acetone:methanol (not shown). This breakdown phenomenon was prevented by using freshly purified acetone. This procedure can be applied to the direct purification of sphingoid bases obtained by acid hydrolysis of complex sphingolipids. Usually, sphingoid bases liberated during acid hydrolysis (8) are purified from the other products of hydrolysis (fatty acids and their methyl esters) by washing the aqueous hydrolysis mixture with a nonpolar solvent like hexane followed by partition with diethyl ether after alkalinization. As shown in Fig. 2, the procedure described in this report allows the efficient recovery of sphingoid bases from hydrolysis mixtures of free ceramides and neutral glycosphingolipids isolated from fresh human melanoma tumors and purified on aminopropyl column chromatography as previously described (9, 10). This is done by directly passing the chloroformic extract of the hydrolysis mixture onto a LC-WCX column preconditioned as described above. The recovery was optimal since no positive spot could be detected by ninhydrin reagent in the chloroform:methanol (9:3, v/v) fraction eluted from the column (not shown). The chromatogram of Fig. 2 shows the presence of a significant proportion of sphingoid bases migrating as sphinganine standard. This observation is interesting since this LCB, which is a rather minor LCB in mammalian
247
and human tissues, has already been reported to be enriched in some cancer types compared to normal tissues (11, 12). This procedure is particularly useful when radioactive determination is required because neither washing protocol nor partition is needed compared with previous methods (8). Up to 1 mg of lipid mixture can be applied onto a 100-mg LC-WCX column and up to 95% of sphingoid bases are efficiently recovered by using this procedure (not shown). The yield of sphingoid bases from hydrolysis mixtures after partitioning with ether is sometimes very incomplete and partitioning needs to be repeated several times. Moreover, this SPE procedure is rapid (20 min is sufficient to prepare the different solvents and to carry out both column conditioning and elutions) and low solvent-consuming. Neutral glycosphingolipids and free LCB can be directly processed for further analysis compared to other preparative methods such as TLC. REFERENCES 1. Lavie, Y., Blusztajn, J. K., and Liscovitch, M. (1994) Formation of endogenous free sphingoid bases in cells induced by changing medium conditions. Biochim. Biophys. Acta 1220, 323-328. 2. Sweeley, C. C., and Moscatelli, E. A. (1959) Qualitative microanalysis and estimation of sphingolipid bases. J. Lipid Res. 1, 40-47. 3. Solfrizzo, M., Avantaggiato, G., and Visconti, A. (1997) Rapid method to determine sphinganine/sphingosine in human and animal urine as a biomarker for fumonisin exposure. J. Chromatogr. B. 692, 87-93. 4. Shephard, G. S., and van der Westhuizen, L. (1998) Liquid chromatographic determination of the sphinganine/sphingosine ratio in serum. J. Chromatogr. B 710, 219-222. 5. Saı¨to, S., and S. Hakomori. (1971) Quantitative determination of total glycosphingolipids from animal cells. J. Lipid Res. 12, 257259. 6. Kannagi, R., Watanabe, K., and S. Hakomori. (1987) Isolation and purification of glycosphingolipids by high-performance liquid chromatography. Methods Enzymol. 138, 3-12. 7. Sambasivarao, K., and McCluer, R. H. (1963) Thin-layer chromatographic separation of sphingosine and related bases. J. Lipid Res. 4, 106-108. 8. Gaver, R. C., and Sweeley, C. C. (1965) Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J. Am. Oil Chem. Soc. 42, 294-298. 9. Bodennec, J., Brichon, G., Portoukalian, J., and Zwingelstein, G. (1999) Improved methods for the simultaneous separation of free diacylglycerol species from ceramides containing phytosphingosine and sphingosine bases with non hydroxy and alpha-hydroxy fatty acids. J. Liq. Chromatogr. Rel. Technol. 22, 14931502. 10. Bodennec, J., Zwingelstein, G., Koul, O., Brichon, G., and Portoukalian, J. (1998) Phytosphingosine biosynthesis differs from sphingosine in fish leukocytes and involves a transfer of methyl groups from [ 3H-methyl]methionine precursor. Biochem. Biophys. Res. Commun. 250, 88-93. 11. Rylova, S. N., Somova, O. G., and Dyatlovitskaya, E. V. (1998) Comparative investigation of sphingoid bases and fatty acids in
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ceramides and sphingomyelins from human ovarian malignant tumors and normal ovary. Biochemistry (Moscow) 63, 1057-1060. 12. Rylova, S. N., Somova, O. G., Zubova, E. S., Dubnik, L. B., Kogtev, L. S., Kozlov, A. M., Alesenko, A. V., and Dyatlovitskaya, E. V. (1999) Content and structure of ceramide and sphingomyelin and sphingomyelinase activity in mouse hepatoma-22. Biochemistry (Moscow) 64, 437-441.
A Sensitive Assay for Phosphoinositide Phosphatases Tomohiko Maehama, Gregory S. Taylor, James T. Slama,* and Jack E. Dixon 1 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606; and *Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43617 Received December 23, 1999
A colorimetric method for the determination of inorganic phosphate employing malachite green has been widely used to quantitate protein phosphatase activity (1–3). This assay is based on the change of absorbance at 620 nm due to formation of a phosphomolybdate– malachite green complex (4, 5) and is able to detect phosphate release from protein phosphatase substrates (1–3). Recently, several phosphoinositide (PI) 2 phosphatases, including PTEN (6 –9) and Sac1p (10), were identified as important modulators of PI-mediated intracellular signaling. Although radiolabeled PIs have been commonly used as substrates in PI phosphatase assays, the radioactivity-based assay system is relatively complicated and time-consuming. It has been difficult to use dye-based methods for PI phosphatase assays, since light scatter from the lipid suspension increases the absorbance at 620 nm, thereby interfering with the colorimetric measurement. Here, we show that removal of lipids by rapid centrifugation dramatically reduces background absorbance, and that this modified dye-based phosphate determination method can be applied to PI phosphatase assays. We also show that N-ethylmaleimide (NEM) irreversibly inhibits the PI phosphatase activity of PTEN and Sac1p and is compatible with the dye-based phosphatase assays. 1 To whom correspondence should be addressed. Fax: (734) 7636492. E-mail: [email protected]. 2 Abbreviations used: PI, phosphoinositide; NEM, N-ethylmaleimide; PIP 3, phosphatidylinositol 3,4,5-trisphosphate; PS, phosphatidylserine; PI(4)P, phosphatidylinositol 4-monophosphate.
Analytical Biochemistry 279, 248 –250 (2000) doi:10.1006/abio.2000.4497 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Materials and Methods Purification of recombinant PTEN. Recombinant human PTEN and a catalytically inactive C124S mutant were prepared as previously described (6) with the following minor modifications. The expression vector pGEX-6P-1 (Amersham–Pharmacia) was used instead of pGEX-KG, and the GST-PTEN fusion protein bound to the glutathione beads was cleaved and released by PreScission Protease (Amersham–Pharmacia) according to the manufacturer’s protocol. PreScission Protease-treated PTEN was eluted from the beads in the supernatant, which was concentrated and stored at ⫺80°C until use. Expression and purification of recombinant Sac1p. A truncated form of Sac1p from Saccharomyces cerevisiae was expressed as a recombinant His-tagged fusion protein in Escherichia coli BL21(DE3) Codon Plus cells (Stratagene). A cDNA fragment encoding residues 1–507 of Sac1p was amplified by polymerase chain reaction from yeast genomic DNA with 5⬘-SacI and 3⬘-XhoI restriction linkers and inserted into the appropriate sites of the pET-21a bacterial expression vector (Novagen). Competent cells were transformed according to the manufacturer’s protocol and plated overnight on 2⫻ YT medium containing 100 g/ml ampicillin and 34 g/ml chloramphenicol. One-liter cultures of 2⫻ YT medium containing antibiotics were inoculated directly from bacterial colonies and grown at 37°C until the OD 600 reached ⬃0.7. Cells were chilled on ice for 20 min, fresh antibiotics added, and protein expression was induced overnight at room temperature by the addition of isopropyl--D-thiogalactopyranoside to a final concentration of 0.4 mM. Following induction, the Sac1p(1–507)-His 6 fusion protein was purified from a bacterial lysate using Ni 2⫹agarose affinity resin (Qiagen). Bacteria from a 1-liter culture were disrupted by sonication in 30 ml of lysis buffer consisting of 50 mM Tris–HCl (pH 8), 300 mM NaCl, 20 mM imidazole–HCl (pH 8), 0.05% 2-mercaptoethanol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml (each) aprotinin, leupeptin, and pepstatin. Triton X-100 was added to 0.5% (v/v) and the crude lysate centrifuged at 18,000g for 20 min at 4°C to remove insoluble materials. The soluble extract was incubated with 3 ml Ni 2⫹-agarose slurry for 2 h at 4°C to bind the His-tagged fusion protein. The Ni 2⫹-agarose beads were then washed 4 ⫻ 5 min with 10 ml lysis buffer containing 0.5% Triton X-100 at 4°C, followed by two 5-min washes in lysis buffer without detergent. The fusion protein was eluted from the Ni 2⫹agarose resin in two washes (1.5 ml each) of lysis buffer containing 200 mM imidazole–HCl (pH 8). The washes were combined and filtered through a 0.2-m pore size syringe filter. Dithiothreitol and glycerol were then added to 2 mM and 25% (v/v), respectively, and the
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native compared to denaturing lysis conditions were applied (Fig. 1). This was confirmed by a more detailed analysis of the time course of tyrosine phosphorylation after addition of CVI or 10% FCS (Fig. 2). Our results clearly show that if a native lysis buffer is used for immunoprecipitation of phosphotyrosinecontaining proteins, the use of a broad spectrum protein kinase inhibitor like genistein is essential to conserve the phosphorylation status of the cell at the time of lysis. In addition, the same lysate could be used for parallel detection of activated Erk2 and phosphotyrosinylated target proteins (Fig. 2). Tyrosine phosphorylation of intracellular proteins induced by diverse extracellular agents is a transient signal, often in the range of only a few minutes. Therefore, during the procedure of protein extraction, three aspects are essential: First, cell lysis must be rapid and quantitative to accurately reveal the signal intensities at different time points. For this purpose, we use liquid nitrogen to freeze the cells immediately before addition of lysis buffer, stopping all intracellular enzymatic reactions as rapidly as possible. As to the lysis conditions, these should be as gentle as possible to retain the antibody binding sites and prevent unspecific background signals, while permitting sufficient solubilization of the protein(s) of interest (10). Both criteria are met by the modified native lysis buffer. Second, the phosphorylation signal at the time of lysis must be “frozen” throughout the immunoprecipitation procedure. This is best accomplished by using a broad spectrum protein tyrosine kinase inhibitor like genistein in addition to inhibitors of proteases and phosphatases. Finally, the affinity and specificity of the anti-phosphotyrosine antibody used for immunoprecipitation is vital to yield optimal results. As established by other groups, RC20 is an antibody of choice for both immunoprecipitation and immunoblotting of phosphotyrosine-containing proteins. Acknowledgments. This project was funded by SFB 366 C5, DFG Schu 646/10-2. D. Schuppan was recipient of a Hermann-und-LillySchilling professorship. We thank Monika Schmid for expert technical assistance.
REFERENCES 1. Atkinson, J. C., Ru¨hl, M., Becker, J., Ackermann, R., and Schuppan, D. (1996) Exp. Cell Res. 228, 283–291. 2. Ru¨hl, M., Sahin, R., Johannsen, M., Somasundaram, R., Manski, D., Riecken, E. O., and Schuppan, D. (1999) J. Biol. Chem. 274, 34361–34368. 3. Ru¨hl, M., Johannsen, M., Atkinson, J., Sahin, E., Somasundaram, R., Manski, D., Riecken, E. O., and Schuppan, D. (1999) Exp. Cell Res. 250, 548 –557. 4. Ignatoski, K. M. W., and Verderame, M. F. (1996) BioTechniques 20, 794 –796. 5. Hardie, D. G. (1995) A Practical Approach (Hardie, D. G., Ed), IRL Press, Oxford.
6. Clark, E. A., and Hynes, R. O. (1996) J. Biol. Chem. 271, 14814 – 14818. 7. Huang, J., Mohammadi, M., Rodrigues, G. A., and Schlessinger, J. (1995) J. Biol. Chem. 270, 5065–5072. 8. Nahas, N., Molski, T. F., Fernandez, G. A., and Sha’afi, R. I. (1996) Biochem. J. 318, 247–253. 9. van Dijk, M. C., Hilkmann, H., and van Blitterswijk, W. J. (1997) Biochem. J. 325, 303–307. 10. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Purification of Free Sphingoid Bases by Solid-Phase Extraction on Weak Cation Exchanger Cartridges Jacques Bodennec,* ,1 Ce´cile Famy,* Ge´rard Brichon,† Georges Zwingelstein,† and Jacques Portoukalian* *Laboratory of Tumor Glycobiology, Faculty of Medicine Lyon-Sud, 69921 Oullins Cedex, France; and †Institute Michel Pacha, University Claude Bernard-Lyon I, 83500 La Seyne Sur Mer, France Received October 21, 1999
Free sphingoid bases such as sphingosine, dihydrosphingosine (sphinganine), and phytosphingosine (4-hydroxysphinganine) are extracted from cells and tissues in mixtures of solvents such as chloroform– methanol (1) and further purified by phase partition under alkaline conditions to be recovered in the organic phase (1, 2). Free sphingoid bases have been purified from neutral lipids by chromatography on small silica gel columns before further separation by HPLC (3, 4). However, by using such preparative techniques, free sphingoid bases are mostly eluted with neutral glycosphingolipids. Moreover it is often difficult to separate these molecules from each other by TLC. Solid-phase extraction (SPE) 2 could be an interesting alternative to separate free sphingoid bases from neutral glycosphingolipids. SPE is a rapid method and each recovered fraction can be directly used for further analysis, bypassing TLC separation. Here we report the use of weak cation SPE cartridges for the separation of free sphingoid bases from neutral glycosphingolipids. The SPE minicolumns are LC-WCX from Supelco (Saint Quentin Fallavier, France) which are 1 To whom correspondence should be addressed. Fax: (33) 478 86 31 48. E-mail: [email protected]. 2 Abbreviations used: SPE, solid-phase extraction; CMH, ceramide monohexosides; CDH, ceramide dihexosides; CTH, ceramide trihexosides; Globo, globosides; LCBs, long chain bases; LC-WCX, weak cation SPE cartridges.
Analytical Biochemistry 279, 245–248 (2000) doi:10.1006/abio.2000.4454 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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cosphingolipid standards were purified from human melanoma tumors according to the procedure of Saı¨to and Hakomori (5) and further fractionated by HPLC to obtain ceramide monohexosides (CMH), ceramide dihexosides (CDH), ceramide trihexosides (CTH), and globosides (Globo) (6). The SPE LC-WCX columns were preconditioned by passing successively 1 ml of hexane, 2 ml of 0.5 N acetic acid in methanol, 4 ml of methanol, and 4 ml of hexane. The lipid mixture (containing 40 g of each sphingoid base and 40 g of each neutral glycosphingolipid) was resuspended in 200 l of chloroform and applied onto the SPE tube placed on a Visiprep vacuum apparatus (Supelco). Pressure was not used before all the applying solvent was fully passed throughout the column. Then, the neutral glycosphingolipids were recovered in the first tube by eluting the column with 3 ml of chloroform:methanol (9:3, v/v) at a flow rate of 0.3 ml/min (see Fig. 1a). With such eluting solvent, the free long chain bases (LCBs) remain on the column (see Fig. 1b). The LCBs were eluted in a second fraction by washing the column with 2 ml of 1 N acetic acid in methanol. The two collected fractions were dried off under nitro-
FIG. 1. Thin-layer chromatography of the different fractions eluted from the preconditioned (see text) 100 mg LC-WCX cartridge. A mixture of neutral glycosphingolipids and free sphingoid bases is loaded on the column. The first fraction (fraction 1) is obtained by eluting the column with 2 ml chloroform:methanol (9:3, v/v). The second fraction (fraction 2) is obtained after elution of the column with 2 ml of 1 N acetic acid in methanol. The two fractions are dried under nitrogen and taken up in a small amount of chloroform: methanol (2:1, v/v). Each fraction is applied by half on two different HPTLC plates. One plate is developed in chloroform:methanol:water (65:25:4, v/v/v) and sprayed with orcinol reagent (a), and the second plate is developed in chloroform:methanol:2 N ammonia (40:10:1, v/v/v) and then visualized with ninhydrin reagent. Std in a: standard mixture of neutral glycosphingolipids CMH, CDH, CTH, and Globo. Std in b: standard mixture of free sphingoid bases: sphingosine, dihydrosphingosine, phytosphingosine. Lane 1, fraction 1; Lane 2, fraction 2.
made of a silica gel matrix (100 mg) bonded with propanoic acid in a sodium salt form. Such type of bonded phase was selected because, under appropriate conditions, the carboxylic acid functional groups on the column should bind to the amino group of free sphingoid bases with a greater relative affinity than to neutral glycosphingolipids, thus allowing separate elution of these compounds. Sphingoid base standards (D-sphingosine, DL-dihydrosphingosine, and phytosphingosine) were from Sigma (Saint Quentin Fallavier, France). Neutral gly-
FIG. 2. HPTLC plate of long-chain bases extracted and purified on LC-WCX cartridge after acid hydrolysis of both free ceramides and neutral glycosphingolipids from fresh human melanoma tumors separately isolated by aminopropyl column chromatography and TLC according to previously published procedures (9, 10). The ceramide spots and neutral glycosphingolipids are scrapped from the plate and hydrolyzed at 70°C in the presence of 1 ml of methanol:water:conc HCl (100:9.4:8.6, v/v/v) during 18 h. After alkalinization to pH 12 with 2 N KOH, the mixture is extracted with 6 ml of chloroform: methanol (2:1, v/v). The chloroformic lower phase is recovered and passed through a preconditioned LC-WCX cartridge (see text). Sphingoid bases are washed and extracted from the LC-WCX column according to the procedure described in this report and an aliquot of fraction 2 is run on HPTLC plate in chloroform:methanol:2 N ammonia (40:10:1, v/v/v). The plate is visualized with ninhydrin reagent. Lane 1, sphingoid bases from hydrolyzed free ceramides isolated from fresh human melanoma tumors; Lane 2, phytosphingosine standard; Lane 3, dihydrosphingosine standard; Lane 4, sphingosine standard; Lane 5, sphingoid bases from hydrolyzed neutral glycosphingolipids purified from fresh human melanoma tumors. The asterisk (*) shows O-methyl derivatives of LCBs.
NOTES & TIPS
gen and taken up in 100 l chloroform:methanol (2:1, v/v) which were separately applied by half on two different HPTLC plates (silica gel 60 from Merck, Darmstadt, Germany) to resolve free LCBs on the one hand and neutral glycosphingolipids on the other hand. The solvent system was chloroform:methanol:2 N ammonia (40:10:1, v/v/v) for LCBs (7) and chloroform:methanol: water (65:25:4, v/v/v) for neutral glycosphingolipids. LCBs on the HPTLC plate were visualized with ninhydrin reagent and neutral glycosphingolipids with orcinol reagent. Figure 1a shows that the neutral glycosphingolipids were fully recovered in the first fraction. No glycosphingolipid could be detected in the second fraction by spraying the HPTLC plate with orcinol reagent, indicating a complete recovery of these lipids in fraction 1. In a same way, it can be seen on Fig. 1b that free sphingoid bases are recovered in the second fraction since the HPTLC plate of the first fraction is clearly ninhydrin-negative. To ensure strong binding, the use of 0.5 N acetic acid in methanol during the preconditioning procedure is required to activate the propanoic acid groups and to remove sodium ions which are present on the column matrix. Resolution of neutral glycosphingolipids from free sphingoid bases was also possible by using acetone: methanol (9:2.4, v/v) instead of chloroform:methanol. However, the solvent mixture containing chloroform was more adequate since free phytosphingosine was sometimes degraded on the column when using acetone:methanol (not shown). This breakdown phenomenon was prevented by using freshly purified acetone. This procedure can be applied to the direct purification of sphingoid bases obtained by acid hydrolysis of complex sphingolipids. Usually, sphingoid bases liberated during acid hydrolysis (8) are purified from the other products of hydrolysis (fatty acids and their methyl esters) by washing the aqueous hydrolysis mixture with a nonpolar solvent like hexane followed by partition with diethyl ether after alkalinization. As shown in Fig. 2, the procedure described in this report allows the efficient recovery of sphingoid bases from hydrolysis mixtures of free ceramides and neutral glycosphingolipids isolated from fresh human melanoma tumors and purified on aminopropyl column chromatography as previously described (9, 10). This is done by directly passing the chloroformic extract of the hydrolysis mixture onto a LC-WCX column preconditioned as described above. The recovery was optimal since no positive spot could be detected by ninhydrin reagent in the chloroform:methanol (9:3, v/v) fraction eluted from the column (not shown). The chromatogram of Fig. 2 shows the presence of a significant proportion of sphingoid bases migrating as sphinganine standard. This observation is interesting since this LCB, which is a rather minor LCB in mammalian
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and human tissues, has already been reported to be enriched in some cancer types compared to normal tissues (11, 12). This procedure is particularly useful when radioactive determination is required because neither washing protocol nor partition is needed compared with previous methods (8). Up to 1 mg of lipid mixture can be applied onto a 100-mg LC-WCX column and up to 95% of sphingoid bases are efficiently recovered by using this procedure (not shown). The yield of sphingoid bases from hydrolysis mixtures after partitioning with ether is sometimes very incomplete and partitioning needs to be repeated several times. Moreover, this SPE procedure is rapid (20 min is sufficient to prepare the different solvents and to carry out both column conditioning and elutions) and low solvent-consuming. Neutral glycosphingolipids and free LCB can be directly processed for further analysis compared to other preparative methods such as TLC. REFERENCES 1. Lavie, Y., Blusztajn, J. K., and Liscovitch, M. (1994) Formation of endogenous free sphingoid bases in cells induced by changing medium conditions. Biochim. Biophys. Acta 1220, 323-328. 2. Sweeley, C. C., and Moscatelli, E. A. (1959) Qualitative microanalysis and estimation of sphingolipid bases. J. Lipid Res. 1, 40-47. 3. Solfrizzo, M., Avantaggiato, G., and Visconti, A. (1997) Rapid method to determine sphinganine/sphingosine in human and animal urine as a biomarker for fumonisin exposure. J. Chromatogr. B. 692, 87-93. 4. Shephard, G. S., and van der Westhuizen, L. (1998) Liquid chromatographic determination of the sphinganine/sphingosine ratio in serum. J. Chromatogr. B 710, 219-222. 5. Saı¨to, S., and S. Hakomori. (1971) Quantitative determination of total glycosphingolipids from animal cells. J. Lipid Res. 12, 257259. 6. Kannagi, R., Watanabe, K., and S. Hakomori. (1987) Isolation and purification of glycosphingolipids by high-performance liquid chromatography. Methods Enzymol. 138, 3-12. 7. Sambasivarao, K., and McCluer, R. H. (1963) Thin-layer chromatographic separation of sphingosine and related bases. J. Lipid Res. 4, 106-108. 8. Gaver, R. C., and Sweeley, C. C. (1965) Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J. Am. Oil Chem. Soc. 42, 294-298. 9. Bodennec, J., Brichon, G., Portoukalian, J., and Zwingelstein, G. (1999) Improved methods for the simultaneous separation of free diacylglycerol species from ceramides containing phytosphingosine and sphingosine bases with non hydroxy and alpha-hydroxy fatty acids. J. Liq. Chromatogr. Rel. Technol. 22, 14931502. 10. Bodennec, J., Zwingelstein, G., Koul, O., Brichon, G., and Portoukalian, J. (1998) Phytosphingosine biosynthesis differs from sphingosine in fish leukocytes and involves a transfer of methyl groups from [ 3H-methyl]methionine precursor. Biochem. Biophys. Res. Commun. 250, 88-93. 11. Rylova, S. N., Somova, O. G., and Dyatlovitskaya, E. V. (1998) Comparative investigation of sphingoid bases and fatty acids in
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ceramides and sphingomyelins from human ovarian malignant tumors and normal ovary. Biochemistry (Moscow) 63, 1057-1060. 12. Rylova, S. N., Somova, O. G., Zubova, E. S., Dubnik, L. B., Kogtev, L. S., Kozlov, A. M., Alesenko, A. V., and Dyatlovitskaya, E. V. (1999) Content and structure of ceramide and sphingomyelin and sphingomyelinase activity in mouse hepatoma-22. Biochemistry (Moscow) 64, 437-441.
A Sensitive Assay for Phosphoinositide Phosphatases Tomohiko Maehama, Gregory S. Taylor, James T. Slama,* and Jack E. Dixon 1 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606; and *Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43617 Received December 23, 1999
A colorimetric method for the determination of inorganic phosphate employing malachite green has been widely used to quantitate protein phosphatase activity (1–3). This assay is based on the change of absorbance at 620 nm due to formation of a phosphomolybdate– malachite green complex (4, 5) and is able to detect phosphate release from protein phosphatase substrates (1–3). Recently, several phosphoinositide (PI) 2 phosphatases, including PTEN (6 –9) and Sac1p (10), were identified as important modulators of PI-mediated intracellular signaling. Although radiolabeled PIs have been commonly used as substrates in PI phosphatase assays, the radioactivity-based assay system is relatively complicated and time-consuming. It has been difficult to use dye-based methods for PI phosphatase assays, since light scatter from the lipid suspension increases the absorbance at 620 nm, thereby interfering with the colorimetric measurement. Here, we show that removal of lipids by rapid centrifugation dramatically reduces background absorbance, and that this modified dye-based phosphate determination method can be applied to PI phosphatase assays. We also show that N-ethylmaleimide (NEM) irreversibly inhibits the PI phosphatase activity of PTEN and Sac1p and is compatible with the dye-based phosphatase assays. 1 To whom correspondence should be addressed. Fax: (734) 7636492. E-mail: [email protected]. 2 Abbreviations used: PI, phosphoinositide; NEM, N-ethylmaleimide; PIP 3, phosphatidylinositol 3,4,5-trisphosphate; PS, phosphatidylserine; PI(4)P, phosphatidylinositol 4-monophosphate.
Analytical Biochemistry 279, 248 –250 (2000) doi:10.1006/abio.2000.4497 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Materials and Methods Purification of recombinant PTEN. Recombinant human PTEN and a catalytically inactive C124S mutant were prepared as previously described (6) with the following minor modifications. The expression vector pGEX-6P-1 (Amersham–Pharmacia) was used instead of pGEX-KG, and the GST-PTEN fusion protein bound to the glutathione beads was cleaved and released by PreScission Protease (Amersham–Pharmacia) according to the manufacturer’s protocol. PreScission Protease-treated PTEN was eluted from the beads in the supernatant, which was concentrated and stored at ⫺80°C until use. Expression and purification of recombinant Sac1p. A truncated form of Sac1p from Saccharomyces cerevisiae was expressed as a recombinant His-tagged fusion protein in Escherichia coli BL21(DE3) Codon Plus cells (Stratagene). A cDNA fragment encoding residues 1–507 of Sac1p was amplified by polymerase chain reaction from yeast genomic DNA with 5⬘-SacI and 3⬘-XhoI restriction linkers and inserted into the appropriate sites of the pET-21a bacterial expression vector (Novagen). Competent cells were transformed according to the manufacturer’s protocol and plated overnight on 2⫻ YT medium containing 100 g/ml ampicillin and 34 g/ml chloramphenicol. One-liter cultures of 2⫻ YT medium containing antibiotics were inoculated directly from bacterial colonies and grown at 37°C until the OD 600 reached ⬃0.7. Cells were chilled on ice for 20 min, fresh antibiotics added, and protein expression was induced overnight at room temperature by the addition of isopropyl--D-thiogalactopyranoside to a final concentration of 0.4 mM. Following induction, the Sac1p(1–507)-His 6 fusion protein was purified from a bacterial lysate using Ni 2⫹agarose affinity resin (Qiagen). Bacteria from a 1-liter culture were disrupted by sonication in 30 ml of lysis buffer consisting of 50 mM Tris–HCl (pH 8), 300 mM NaCl, 20 mM imidazole–HCl (pH 8), 0.05% 2-mercaptoethanol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml (each) aprotinin, leupeptin, and pepstatin. Triton X-100 was added to 0.5% (v/v) and the crude lysate centrifuged at 18,000g for 20 min at 4°C to remove insoluble materials. The soluble extract was incubated with 3 ml Ni 2⫹-agarose slurry for 2 h at 4°C to bind the His-tagged fusion protein. The Ni 2⫹-agarose beads were then washed 4 ⫻ 5 min with 10 ml lysis buffer containing 0.5% Triton X-100 at 4°C, followed by two 5-min washes in lysis buffer without detergent. The fusion protein was eluted from the Ni 2⫹agarose resin in two washes (1.5 ml each) of lysis buffer containing 200 mM imidazole–HCl (pH 8). The washes were combined and filtered through a 0.2-m pore size syringe filter. Dithiothreitol and glycerol were then added to 2 mM and 25% (v/v), respectively, and the