ANALYTICAL
BIOCHEMISTRY
75, 664-667
(1976)
Separation of Mannosylretinylphosphate mannosylphosphate by Solvent
from DolichylExtraction
Extraction and purification of mannosylretinylphosphate (MRP) from liver membranes has been achieved (l-3) using the lower phase of the Folch extraction procedure (4). Moreover MRP is separated from dolichylmannosylphosphate (DMP) by chromatography on thin layers of silica gel in chloroform (60):methanol (25):water (4), a solvent first developed by Tkacz et al. (5). We have reported a modification of the Folch extraction procedure which allows the differential solvent extraction of DMP and MRP from the lower organic phase (3), but MRP obtained by this procedure contains some DMP. Since MRP is much more hydrophylic than DMP we investigated the possibility that some MRP is in the upper phase. Here we report that 70% or more of the MRP is in the upper phase while all of the DMP is in the lower organic phase. All operations were conducted in red light. Rat liver membranes were prepared as previously described (2,3) from normal rats, and incubations were run in the presence or absence of retinylphosphate (RP) obtained as previously described (3). The incubation mixture contained: 4 mg of normal rat liver enzyme protein suspended in 0.1 ml of TMK (6) buffer, pH 7.6; 20 ~1 of a 0.025 M solution of EDTA; 20 ~1 of 0.3 M Tris-HCl buffer, pH 8; 20 4 of 0.1 M MnCl,; 10 ~1 of a solution of ATPcontaining 22 mg/ml; 20 ~1 of water containing 2 &i of [U-14C]guanosine diphosphatemannose, specific activity of 221 mCi/mmol. The final pH was 7.6. The equivalent of 0.2 ml of rat liver lipid extract obtained from 0.6 mg of enzyme membrane protein was dried and redissolved in 10 ~1 of DMSO with or without RP. Three incubations containing 0,20, and 40 pg of RP were run for 30 min at 37°C and the reaction was stopped with 3 ml (15 volumes) of chloroform (2):methanol (1) and 0.6 ml (four volumes) of 0.9% NaCl. The upper and lower phases were separated by low speed centrifugation, and the lower phase was washed with 400 ~1 of 0.9% saline, as previously described (3); the resulting lower organic phase contains mostly DMP (3). The combined aqueous phases and washing were extracted again with 2 ml of chloroform (2):methanol (1) which represents 10 times the volume of the original incubation. The resulting lower organic phase contains mostly retinylphosphatemannose. This lower phase was combined with the first lower phase obtained in the first extraction, dried, and redissolved in 0.5 ml of chloroform (2):methanol (l), and 50 ~1 was chromatographed on thin layers of silica gel in solvent A. The dry plates were viewed under a uv lamp to 664 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
665
cm
FIG. 1. Separation of MRP from DMP. (A), Chromatography on thin layers of silica gel in solvent A of one-tenth of the total lower organic extract. The incubation was run as described in the text with 4Opg of synthetic RP. The position of RP is indicated by the black spot. MRP has&O.3 and DMP hasR,0.61. (B), Chromatography on thin layer of silicagel in solvent A of one-tenth of the total upper phase. The incubation was run as described in the text with 40 pg of synthetic RP. The position of RP is marked by the black spot. MRP has R,0.3. No DMP is present.
locate standard RP and scraped with an automatic plate scraper. The scraped gel was suspended in 0.1 ml of 9% methanol for 1 hr prior to adding 15 ml of a solution of 5 g of 2,5-diphenyloxazone and 50 mg of 1,4-bis-[2-(5-phenyloxazolyl)] benzene in 1000 ml of toluene. Counting efficiency was 66%. The upper phase was dried under a stream of nitrogen, dissolved in 2 ml of 9% methanol, and applied to a column (1 x 5 cm) of DEAE-celluloseacetate as described (7). The column was eluted first with 250 ml of 99% methanol to remove all unwanted products and then with 25 ml of 0.050 M ammonium acetate in 9% methanol, which elutes both MRP and DMP (3). The eluate was dried under nitrogen and redissolved in 2.5 ml of chloroform (2):methanol (l), and 0.25 ml was used for chromatography on thin layers of silica gel in solvent A, along with standard RP. Scraping and assaying of radioactivity was the same as for the organic lower phases. A typical separation of MRP (R,0.3) from DMP (R,0.61) is shown in Fig. lA, which illustrates the analysis of the organic lower phase of the
666
SHORT COMMUNICATIONS
FIG. 2. Effect of RP on the synthesis of MRP and DMP. Stimulation of MRP synthesis by RP in the lower (0 0) and upper (A A) phase is shown. No effect on DMP (0 0) synthesis is observed with normal enzyme at these concentrations of RP.
incubation containing 40 lug of RP. Figure 1B shows the result of chromatography of the upper phase obtained from the same incubation and which contains no DMP. The distribution of MRP is 30% in the organic lower phase and 70% in the upper phase. Stimulation of MRP synthesis by RP in the lower and in the upper phase is shown in Fig. 2. Clearly RP does not affect the synthesis of DMP when normal liver membranes are used (fig. 2), whereas inhibition of DMP synthesis by RP was found with vitamin A-deficient rat liver membranes (3). We conclude that 70% of MRP free of DMP is obtained in the upper phase of the described extraction procedure. In the absence of exogenous RP, MRP represents about 10% of the total mannolipid synthesized by rat liver membrane. This becomes totally soluble in the upper phase, if more than one wash with theoretical upper phase (4) is used. REFERENCES 1. De Luca, L. M., Rosso, G. C., and Wolf, G. (1970)Eiochem. Biophys. Res. Commun. 41, 61.5-620.
De Luca, L. M., Maestri, N., Rosso, G. C., and Wolf, G. (1973) J. Biol. Chem. 248, 641448. 3. Rosso, G. C., De Luca, L. M., Warren, C. D., and Wolf, G. (1975) J. Lipid Res. 16, 2.
235-243.
SHORT COMMUNICATIONS
667
4. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509. 5. Tkacz, J. C., Herscovics, A., Warren, C. D., and Jeanloz, R. W. (1974)J. Biol. Chem.
249,6372-6381. 6. De Luca, L. M., Little. E. P., and Wolf, G. (1%9) J. Biol. Chem. 244, 701-708. 7. Frot-Coutaz, J. P., Silverman-Jones, C. S., and De Luca, L. M., 1976, J. Lipid Res. 17, 220-230. CAROLS. SILVERMAN-JONES JACQUESP. FROT-COUTAZ LUIGI M. DE LUCA Experimental Pathology Branch National Cancer Institute Bethesda, Maryland 20014 Received March 3, 1976; accepted June 10, 1976
BIOCHEMISTRY
75, 664-667
(1976)
Separation of Mannosylretinylphosphate mannosylphosphate by Solvent
from DolichylExtraction
Extraction and purification of mannosylretinylphosphate (MRP) from liver membranes has been achieved (l-3) using the lower phase of the Folch extraction procedure (4). Moreover MRP is separated from dolichylmannosylphosphate (DMP) by chromatography on thin layers of silica gel in chloroform (60):methanol (25):water (4), a solvent first developed by Tkacz et al. (5). We have reported a modification of the Folch extraction procedure which allows the differential solvent extraction of DMP and MRP from the lower organic phase (3), but MRP obtained by this procedure contains some DMP. Since MRP is much more hydrophylic than DMP we investigated the possibility that some MRP is in the upper phase. Here we report that 70% or more of the MRP is in the upper phase while all of the DMP is in the lower organic phase. All operations were conducted in red light. Rat liver membranes were prepared as previously described (2,3) from normal rats, and incubations were run in the presence or absence of retinylphosphate (RP) obtained as previously described (3). The incubation mixture contained: 4 mg of normal rat liver enzyme protein suspended in 0.1 ml of TMK (6) buffer, pH 7.6; 20 ~1 of a 0.025 M solution of EDTA; 20 ~1 of 0.3 M Tris-HCl buffer, pH 8; 20 4 of 0.1 M MnCl,; 10 ~1 of a solution of ATPcontaining 22 mg/ml; 20 ~1 of water containing 2 &i of [U-14C]guanosine diphosphatemannose, specific activity of 221 mCi/mmol. The final pH was 7.6. The equivalent of 0.2 ml of rat liver lipid extract obtained from 0.6 mg of enzyme membrane protein was dried and redissolved in 10 ~1 of DMSO with or without RP. Three incubations containing 0,20, and 40 pg of RP were run for 30 min at 37°C and the reaction was stopped with 3 ml (15 volumes) of chloroform (2):methanol (1) and 0.6 ml (four volumes) of 0.9% NaCl. The upper and lower phases were separated by low speed centrifugation, and the lower phase was washed with 400 ~1 of 0.9% saline, as previously described (3); the resulting lower organic phase contains mostly DMP (3). The combined aqueous phases and washing were extracted again with 2 ml of chloroform (2):methanol (1) which represents 10 times the volume of the original incubation. The resulting lower organic phase contains mostly retinylphosphatemannose. This lower phase was combined with the first lower phase obtained in the first extraction, dried, and redissolved in 0.5 ml of chloroform (2):methanol (l), and 50 ~1 was chromatographed on thin layers of silica gel in solvent A. The dry plates were viewed under a uv lamp to 664 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
665
cm
FIG. 1. Separation of MRP from DMP. (A), Chromatography on thin layers of silica gel in solvent A of one-tenth of the total lower organic extract. The incubation was run as described in the text with 4Opg of synthetic RP. The position of RP is indicated by the black spot. MRP has&O.3 and DMP hasR,0.61. (B), Chromatography on thin layer of silicagel in solvent A of one-tenth of the total upper phase. The incubation was run as described in the text with 40 pg of synthetic RP. The position of RP is marked by the black spot. MRP has R,0.3. No DMP is present.
locate standard RP and scraped with an automatic plate scraper. The scraped gel was suspended in 0.1 ml of 9% methanol for 1 hr prior to adding 15 ml of a solution of 5 g of 2,5-diphenyloxazone and 50 mg of 1,4-bis-[2-(5-phenyloxazolyl)] benzene in 1000 ml of toluene. Counting efficiency was 66%. The upper phase was dried under a stream of nitrogen, dissolved in 2 ml of 9% methanol, and applied to a column (1 x 5 cm) of DEAE-celluloseacetate as described (7). The column was eluted first with 250 ml of 99% methanol to remove all unwanted products and then with 25 ml of 0.050 M ammonium acetate in 9% methanol, which elutes both MRP and DMP (3). The eluate was dried under nitrogen and redissolved in 2.5 ml of chloroform (2):methanol (l), and 0.25 ml was used for chromatography on thin layers of silica gel in solvent A, along with standard RP. Scraping and assaying of radioactivity was the same as for the organic lower phases. A typical separation of MRP (R,0.3) from DMP (R,0.61) is shown in Fig. lA, which illustrates the analysis of the organic lower phase of the
666
SHORT COMMUNICATIONS
FIG. 2. Effect of RP on the synthesis of MRP and DMP. Stimulation of MRP synthesis by RP in the lower (0 0) and upper (A A) phase is shown. No effect on DMP (0 0) synthesis is observed with normal enzyme at these concentrations of RP.
incubation containing 40 lug of RP. Figure 1B shows the result of chromatography of the upper phase obtained from the same incubation and which contains no DMP. The distribution of MRP is 30% in the organic lower phase and 70% in the upper phase. Stimulation of MRP synthesis by RP in the lower and in the upper phase is shown in Fig. 2. Clearly RP does not affect the synthesis of DMP when normal liver membranes are used (fig. 2), whereas inhibition of DMP synthesis by RP was found with vitamin A-deficient rat liver membranes (3). We conclude that 70% of MRP free of DMP is obtained in the upper phase of the described extraction procedure. In the absence of exogenous RP, MRP represents about 10% of the total mannolipid synthesized by rat liver membrane. This becomes totally soluble in the upper phase, if more than one wash with theoretical upper phase (4) is used. REFERENCES 1. De Luca, L. M., Rosso, G. C., and Wolf, G. (1970)Eiochem. Biophys. Res. Commun. 41, 61.5-620.
De Luca, L. M., Maestri, N., Rosso, G. C., and Wolf, G. (1973) J. Biol. Chem. 248, 641448. 3. Rosso, G. C., De Luca, L. M., Warren, C. D., and Wolf, G. (1975) J. Lipid Res. 16, 2.
235-243.
SHORT COMMUNICATIONS
667
4. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509. 5. Tkacz, J. C., Herscovics, A., Warren, C. D., and Jeanloz, R. W. (1974)J. Biol. Chem.
249,6372-6381. 6. De Luca, L. M., Little. E. P., and Wolf, G. (1%9) J. Biol. Chem. 244, 701-708. 7. Frot-Coutaz, J. P., Silverman-Jones, C. S., and De Luca, L. M., 1976, J. Lipid Res. 17, 220-230. CAROLS. SILVERMAN-JONES JACQUESP. FROT-COUTAZ LUIGI M. DE LUCA Experimental Pathology Branch National Cancer Institute Bethesda, Maryland 20014 Received March 3, 1976; accepted June 10, 1976
