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Determination of phospholipid composition of RNA tumor viruses by 32P labeling of infected cell cultures
ANALYTICAL
47, 614-619
BIOCHEMISTRY
Determination Viruses
(1972)
of Phospholipid by “P
.JARIES
Labeling
I’. QI’IGLEY, MIRIAM
Composition of Infected
of RNA Cell
DANIEL B. RIFKIN, H. EINHORN
Received Octohcr
Tumor
Cultures AND
27. 1971
An important substructure of many animal viruses, including the RNA tumor viruses, is the viral envelope, whose major lipid component is pliospholil~id. Many of these viruses arc purified from cells in culture, and the quantities of virus and membrane that can ordinarily be harvested from each Petri dish are in the microgram range. Since phospholipid analysis by thin-layer chromatography may require milligram quantities of starting material, large volumes of culture fluids and/or infected cells arc needed for detailed studies of viral and membrane l~l~osl~holil~id. As a basis for investigating the viral and cellular lipids in cultures infected by Rous sarcoma virus and other enveloped viruses, it appeared desirable to simplify the rquirements for starting materials. The starting point for the present method is the observation that cells in culture do not incorporate either intact phospholipid molecules or the lipid phosphorus in serum int’o cellular or viral phospholipids (1). This makes it possible to grow cells for long periods in ‘+(2Pmedia of known, constant specific radioactivity and permits l~hospholipid analysis of viral and cellular membranes to be performed accurately on microgram amounts of material. METHODS
Cells. Primary cultures of chick embryo fibroblasts (CEF) were prepared from 11-12 day embryos as previously described (2). These were maintained and propagated in Eagle’s minimal medium supplemented with 105% fetal bovine serum. Unless otherwise noted, transformed cells were infected at least 6 days before use. VLl’us. The Rous sarcoma virus (RSV) strain used was a high-titer variant of Schmidt-Ruppin virus that was purified as previously described (1). Cell culture fluid is the starting material for virus purification. Total Cell Menabranes. Cultures of CEF were washed with cold iso614
@ 1972
by
AcademicPress,Inc.
PHOSPHOLIPID
OF
RNA
TUMOR
VIRUSEP
615
tonic saline and scraped from the culture dish with a rubber policeman. The cells were resuspended in cold isotonic saline and washed twice by centrifugation at 1OOOg for 5 min. The pellet was resuspended in 10 vol Tris (50 mM)/EDTA (1 1~~111) buffer, pH 7.2, and homogenized at O-4” in a tight-fitting Dounre homogenizr>r until all of the cells were broken. The homogcnatc was centrifuged at 30&5OOg for 5 min to remove nuclei and any remaining unbroken cells, and the supernatant was cent.rifuged at 100,OOOgfor 90 min to pellet all the cell membranes. Phospholipid Analysis. Total cell membranes and purified virions were extracted with chloroform/methanol according to the procedure of Folch et al. (3). The extract was washed with ‘/j vol 0.05 M KC1 and again with ‘kj vol chloroform/methanol/KCl, 3:48:47. The washed organic extract was taken to dryness under nitrogen and the lipids redissolved in chloroform/methanol, 2:l. The isolated lipids, labeled with 3ZP,were analyzed by thin-layer chromatography (TLC) on silica gel H plates, prepared according to Skipski et al. (4) or OD precoatcd silica gel plates (Eastman). The plates were run for 90 min in a solvent system of chloroform/methanol/glacial acetic acid/water, 25: 1,5:4:2. All lipids were detected by st,aining with iodine vapor, phospholipids by staining with a molybdate spray (5)) and amino lipids by spraying with 0.57, ninhydrin in butnnol. The zones containing phospholipid were then scraped from the thin-layer plates, and analyzed for phosphorus according to the method of Bartlett (6) and for Y? in a scintillation counter. RESULTS
Cultures of RSV-transformed CEF were grown in media containing aZP (I &i/ml, 2 &i/,molej for 4 days with daily replacement of medium. After 2 days (approximately 3 cell divisions) cellular phospholipids had reached constant. specific activity (Fig. 1). At various times during the labeling period the phospholipids were analyzed by thinlayer chromatography and the percentage of the total 32P radioactivity in each phospholipid species was calculated. Figure 2 indicates that the major cellular phospholipids maintained a constant percent#ageof 32P after 2-3 days of labeling. To establish whether these radioactivity percentages represent the actual phospholipid composition in membranes, the total cellular membrane phospholipids were examined by quantit’ative thin-layer chromatography after labeling the cells with 3ZP for 4 days. The percentage composition of individual phospholipids was determined both by I*P counting and by the chemical determination of lipid phosphorus. The phospholipids of RSV, purified from these same cultures of CEF, were also analyzed in the same way.
QCIGLEY,
OVI
RIFKIN,
IO
’
Ah-D
’
30
’
/
50
Time
EIXHORN
I
I
70
I
’
90
’
(hr)
1. Incorporation of 32P into cellular phospholipids. Cultures of RSV-transformed CEF were labeled with “P (1 &i/ml, 2 &i/pmole) for 4 days with daily replacement of media. At, indicated times after addition of 32P the cells were were extracted as described scraped from the culture dia!lr J and the phospholipids in “Methods.” The rxtt,act was analyzed for lipid phosphorus (6) and an aliyuot was counted in liquid scintillation fluid (7). The specific activity (cpm/pmolc lipid phosphorus) was calculated for each time point. FIG.
The data in Fig. 3 are given as the representation of thin-layer chromatograms showing tlic rclativc migration of the incliridual cellular and viral phospholipids. The percentage of the total contributed hy each species and detcrmincd hy hot11 analytical methods is given hy the bar graphs at, the podtion of the rcqwctivc locations on the chromatogram. The results tlcmonstrntc that after a 4 day labeling period the specific radioactivity of phoq~lioruc is the s:Lmc in all species of cellular and
Time
(hr)
FIG. 2. Change in relative percentage of 3’P in each of the major cellular phospholipids. 3’P phospholipids were prepared from CEF as described in Fig. 1. At the indicated times the phospholipids were separated by TLC and the per cent of total radioactivity in each phospholipid species was calculated. Abbreviations: sph, sphingomyelin; PI, phosphatidyl inositol ; PS, phosphatidyl serine; PE, phosphatidyl ethanolamine ; PC, phosphatidyl choline.
PHOSPHOLIPID
OF
RX.4
TUMOR
VIRUSES
617
IOrk% Orlgln
sph
PC
PI PS
PE
Front
FIG. 3. Thin-layer chromatogram representing per cent composition of phospholipids extracted from ROW sarcoma virus (RSV) and chick embryo fibroblasts (CEF) determined by two analytical methods. Cultures of RSV-transformed chick 4 pCi/pmole) with daily reembryo fibroblasts were labeled with “P (2 $X/ml, placement of media. After 4 days. the virus Ivas harvested and purified and the t,otal cell membranes were prepared as described in “Methods.” The phospholipids were extracted from the virus and ~11 membranes. Thr lipid extract was spotted on a silica gel H plate, chromatogrnphed, and stained, and each of the separated phospholipid species was scraped from the plates and extracted (4). Each extracat was analyzed for lipid phosphorus (5) and an aliquot was also counted in liquid scintillation fluid. The phospholipid (01 “P) percentage. based on total recovev, was calculated for each separated phospholipid specks. The results arc the average of 3 separate experiments in which the recovery ranged from 75 to 9Or/u. The positions of phospholipid standards appear at the bottom of the figure. The lower bar graph represents the values for RSV and the upprr graph thcx values for CEF. Abbreviations as in Fig. 2.
viral phospholipids. Therefore, the chemically determined distribution of lipid phosphorus in the cellular and kzd I~hosI)holipid fractions is the same as t,hat obtained from measurements of incorporated radioactivity in t,he same fractions. Figure 4 represents a further improvement of the method. A few micrograms of 32P-labeled CEF phospholipids were spotted on a commercial (Eastman) thin-layer sheet, which were then chromatographed along with standard phospholipide, dried, stained, and cut in 5 mm slices. Each slice was counted in toluene/Liquifluor scintillation fluid; the observed percentage of the total radioactivity is given under each peak. These values are, within experimental error, identical with the percentage composition determined by the chemical met’hod (Fig. 3). This procedure is much less time consuming and less cumbersome than microchemical analysis of phospholipids which must be extracted from scrapings of silica gel chromatography plates. In addition, the method enables
618
QUIGLEY,
RIFKIN,
cm
AND
PC
vh
1500
EINHORK
PI PS
PE
h
2
6
IO
14
18
22
SI ice number extracted from chick FIG. 4. Thin-layer chromatography of 3’P phospholipids “‘P-labeled WV-transformed embryo fibroblasts. The total cell membranes from chick embryo fibroblasts were prepared. The phospholipids were extracted and spotted, along with standard phospholipids used as unlabeled carrier, on an East-
man thin-layer silica gel sheet, and chromatographed in a solvent system of chloroform/methanol/glacial acetic acid/water. 25: 15:4:2. The chromatogram was removed from the solvent system after 7&90 min (10-11 cm), stained with iodine, and cut in 5 mm slices. Each slice was counted in a toluene/Liquiflour scintillation fluid. The percentage of 32P for each phospholipid species on the chromatogram was calculated, base,d on total recovery, and the resulting values are given under each peak. The positions of the phospholipid standards appear at the top of the figure. Abbreviations as in Fig. 2. one to determine the composition of viral phospholipid with small amounts of starting material and without sacrificing accuracy or precision. Approximately 4 pg of purified RSV phospholipids, labeled with 32P, can be obtained from the cell culture fluid of a single Petri dish and contain enough radioactivity (22000 cpm) to perform the analysis, whereas microchemical determination of phospholipid composition would require 50-100 times more material. DISCUSSIOPI;
The present study demonstrates that, the phospholipid composition of tumor viruses can be determined by analyzing the virus released by a relatively small number of 3ZP-labeled infected cells (1 X 107). The validity of this method is demonstrated by the fact that cells labeled with 32P, as t,he precursor of phospholipid phosphorus, reach a steady state in the turnover of membrane phospholipid (Figs. 1 and 2). During this steady state the specific activity of the lipid phosphorus is the same for each of the individual phospholipid species, and therefore the distribution of the radioactivity in each species of phospholipids represents the actual distribution of the given phospholipid in the membrane (Fig. 3). Since the cells are growing and increasing in mass, and if the inorganic phosphate in the medium is the sole source of phosphorus for cellular components, the specific radioactivity of the total cellular phosphorus,
RNA
PHOSPHOLIPID
OF RNA KMOR
619
VIRGSF,s
and of each of the phosphorus-containing molecules, must asymptotically approach the specific radioactivity of the sole phosphorus precursor, provided the value of the latter is kept constant. The data presented here show that this expectation is fulfilled. ACKNOWLEDGMEiYTS The authors gratefully acknowledge the skilled technical assistance Janice M. Herbert and Miss Linda Steiner. J.P.Q. is a Leukemia Society of America Fellow. D.B.R.. is a Jane Coffin Childs Memorial Fund Fellow. The work was supported by a grant from the American Cancer Society
of
Miss
E-478.
REFERESCES 1. 2. 3. 4. 5. 6. 7.
QUIGLEY. J. P., RIFKIN, D. B., AND REICH, E.. TEMIN, H. M., Int. J. Cancer 3, 273 (1968). FOLCH, J., LEES, M.. AND SLOAN-STASLEY, J. SKIPSKI, V. P., PETERSON, R.. F., ASD BARCLAY, DITTMER, J. ASD LESTER, R., J. Lipid. Res. 5, BARTLETT, P., J. Biol. Chem. 234, 466 (1959). BRAY, G. A., Anal. Biochem. 1, 279 (1960).
Virology
46, 106 (1971).
B., J. Viol. Chem. 226, 497 (195 M., Biochem. J. 90, 374 (1964). 126 (1964).
‘I.
47, 614-619
BIOCHEMISTRY
Determination Viruses
(1972)
of Phospholipid by “P
.JARIES
Labeling
I’. QI’IGLEY, MIRIAM
Composition of Infected
of RNA Cell
DANIEL B. RIFKIN, H. EINHORN
Received Octohcr
Tumor
Cultures AND
27. 1971
An important substructure of many animal viruses, including the RNA tumor viruses, is the viral envelope, whose major lipid component is pliospholil~id. Many of these viruses arc purified from cells in culture, and the quantities of virus and membrane that can ordinarily be harvested from each Petri dish are in the microgram range. Since phospholipid analysis by thin-layer chromatography may require milligram quantities of starting material, large volumes of culture fluids and/or infected cells arc needed for detailed studies of viral and membrane l~l~osl~holil~id. As a basis for investigating the viral and cellular lipids in cultures infected by Rous sarcoma virus and other enveloped viruses, it appeared desirable to simplify the rquirements for starting materials. The starting point for the present method is the observation that cells in culture do not incorporate either intact phospholipid molecules or the lipid phosphorus in serum int’o cellular or viral phospholipids (1). This makes it possible to grow cells for long periods in ‘+(2Pmedia of known, constant specific radioactivity and permits l~hospholipid analysis of viral and cellular membranes to be performed accurately on microgram amounts of material. METHODS
Cells. Primary cultures of chick embryo fibroblasts (CEF) were prepared from 11-12 day embryos as previously described (2). These were maintained and propagated in Eagle’s minimal medium supplemented with 105% fetal bovine serum. Unless otherwise noted, transformed cells were infected at least 6 days before use. VLl’us. The Rous sarcoma virus (RSV) strain used was a high-titer variant of Schmidt-Ruppin virus that was purified as previously described (1). Cell culture fluid is the starting material for virus purification. Total Cell Menabranes. Cultures of CEF were washed with cold iso614
@ 1972
by
AcademicPress,Inc.
PHOSPHOLIPID
OF
RNA
TUMOR
VIRUSEP
615
tonic saline and scraped from the culture dish with a rubber policeman. The cells were resuspended in cold isotonic saline and washed twice by centrifugation at 1OOOg for 5 min. The pellet was resuspended in 10 vol Tris (50 mM)/EDTA (1 1~~111) buffer, pH 7.2, and homogenized at O-4” in a tight-fitting Dounre homogenizr>r until all of the cells were broken. The homogcnatc was centrifuged at 30&5OOg for 5 min to remove nuclei and any remaining unbroken cells, and the supernatant was cent.rifuged at 100,OOOgfor 90 min to pellet all the cell membranes. Phospholipid Analysis. Total cell membranes and purified virions were extracted with chloroform/methanol according to the procedure of Folch et al. (3). The extract was washed with ‘/j vol 0.05 M KC1 and again with ‘kj vol chloroform/methanol/KCl, 3:48:47. The washed organic extract was taken to dryness under nitrogen and the lipids redissolved in chloroform/methanol, 2:l. The isolated lipids, labeled with 3ZP,were analyzed by thin-layer chromatography (TLC) on silica gel H plates, prepared according to Skipski et al. (4) or OD precoatcd silica gel plates (Eastman). The plates were run for 90 min in a solvent system of chloroform/methanol/glacial acetic acid/water, 25: 1,5:4:2. All lipids were detected by st,aining with iodine vapor, phospholipids by staining with a molybdate spray (5)) and amino lipids by spraying with 0.57, ninhydrin in butnnol. The zones containing phospholipid were then scraped from the thin-layer plates, and analyzed for phosphorus according to the method of Bartlett (6) and for Y? in a scintillation counter. RESULTS
Cultures of RSV-transformed CEF were grown in media containing aZP (I &i/ml, 2 &i/,molej for 4 days with daily replacement of medium. After 2 days (approximately 3 cell divisions) cellular phospholipids had reached constant. specific activity (Fig. 1). At various times during the labeling period the phospholipids were analyzed by thinlayer chromatography and the percentage of the total 32P radioactivity in each phospholipid species was calculated. Figure 2 indicates that the major cellular phospholipids maintained a constant percent#ageof 32P after 2-3 days of labeling. To establish whether these radioactivity percentages represent the actual phospholipid composition in membranes, the total cellular membrane phospholipids were examined by quantit’ative thin-layer chromatography after labeling the cells with 3ZP for 4 days. The percentage composition of individual phospholipids was determined both by I*P counting and by the chemical determination of lipid phosphorus. The phospholipids of RSV, purified from these same cultures of CEF, were also analyzed in the same way.
QCIGLEY,
OVI
RIFKIN,
IO
’
Ah-D
’
30
’
/
50
Time
EIXHORN
I
I
70
I
’
90
’
(hr)
1. Incorporation of 32P into cellular phospholipids. Cultures of RSV-transformed CEF were labeled with “P (1 &i/ml, 2 &i/pmole) for 4 days with daily replacement of media. At, indicated times after addition of 32P the cells were were extracted as described scraped from the culture dia!lr J and the phospholipids in “Methods.” The rxtt,act was analyzed for lipid phosphorus (6) and an aliyuot was counted in liquid scintillation fluid (7). The specific activity (cpm/pmolc lipid phosphorus) was calculated for each time point. FIG.
The data in Fig. 3 are given as the representation of thin-layer chromatograms showing tlic rclativc migration of the incliridual cellular and viral phospholipids. The percentage of the total contributed hy each species and detcrmincd hy hot11 analytical methods is given hy the bar graphs at, the podtion of the rcqwctivc locations on the chromatogram. The results tlcmonstrntc that after a 4 day labeling period the specific radioactivity of phoq~lioruc is the s:Lmc in all species of cellular and
Time
(hr)
FIG. 2. Change in relative percentage of 3’P in each of the major cellular phospholipids. 3’P phospholipids were prepared from CEF as described in Fig. 1. At the indicated times the phospholipids were separated by TLC and the per cent of total radioactivity in each phospholipid species was calculated. Abbreviations: sph, sphingomyelin; PI, phosphatidyl inositol ; PS, phosphatidyl serine; PE, phosphatidyl ethanolamine ; PC, phosphatidyl choline.
PHOSPHOLIPID
OF
RX.4
TUMOR
VIRUSES
617
IOrk% Orlgln
sph
PC
PI PS
PE
Front
FIG. 3. Thin-layer chromatogram representing per cent composition of phospholipids extracted from ROW sarcoma virus (RSV) and chick embryo fibroblasts (CEF) determined by two analytical methods. Cultures of RSV-transformed chick 4 pCi/pmole) with daily reembryo fibroblasts were labeled with “P (2 $X/ml, placement of media. After 4 days. the virus Ivas harvested and purified and the t,otal cell membranes were prepared as described in “Methods.” The phospholipids were extracted from the virus and ~11 membranes. Thr lipid extract was spotted on a silica gel H plate, chromatogrnphed, and stained, and each of the separated phospholipid species was scraped from the plates and extracted (4). Each extracat was analyzed for lipid phosphorus (5) and an aliquot was also counted in liquid scintillation fluid. The phospholipid (01 “P) percentage. based on total recovev, was calculated for each separated phospholipid specks. The results arc the average of 3 separate experiments in which the recovery ranged from 75 to 9Or/u. The positions of phospholipid standards appear at the bottom of the figure. The lower bar graph represents the values for RSV and the upprr graph thcx values for CEF. Abbreviations as in Fig. 2.
viral phospholipids. Therefore, the chemically determined distribution of lipid phosphorus in the cellular and kzd I~hosI)holipid fractions is the same as t,hat obtained from measurements of incorporated radioactivity in t,he same fractions. Figure 4 represents a further improvement of the method. A few micrograms of 32P-labeled CEF phospholipids were spotted on a commercial (Eastman) thin-layer sheet, which were then chromatographed along with standard phospholipide, dried, stained, and cut in 5 mm slices. Each slice was counted in toluene/Liquifluor scintillation fluid; the observed percentage of the total radioactivity is given under each peak. These values are, within experimental error, identical with the percentage composition determined by the chemical met’hod (Fig. 3). This procedure is much less time consuming and less cumbersome than microchemical analysis of phospholipids which must be extracted from scrapings of silica gel chromatography plates. In addition, the method enables
618
QUIGLEY,
RIFKIN,
cm
AND
PC
vh
1500
EINHORK
PI PS
PE
h
2
6
IO
14
18
22
SI ice number extracted from chick FIG. 4. Thin-layer chromatography of 3’P phospholipids “‘P-labeled WV-transformed embryo fibroblasts. The total cell membranes from chick embryo fibroblasts were prepared. The phospholipids were extracted and spotted, along with standard phospholipids used as unlabeled carrier, on an East-
man thin-layer silica gel sheet, and chromatographed in a solvent system of chloroform/methanol/glacial acetic acid/water. 25: 15:4:2. The chromatogram was removed from the solvent system after 7&90 min (10-11 cm), stained with iodine, and cut in 5 mm slices. Each slice was counted in a toluene/Liquiflour scintillation fluid. The percentage of 32P for each phospholipid species on the chromatogram was calculated, base,d on total recovery, and the resulting values are given under each peak. The positions of the phospholipid standards appear at the top of the figure. Abbreviations as in Fig. 2. one to determine the composition of viral phospholipid with small amounts of starting material and without sacrificing accuracy or precision. Approximately 4 pg of purified RSV phospholipids, labeled with 32P, can be obtained from the cell culture fluid of a single Petri dish and contain enough radioactivity (22000 cpm) to perform the analysis, whereas microchemical determination of phospholipid composition would require 50-100 times more material. DISCUSSIOPI;
The present study demonstrates that, the phospholipid composition of tumor viruses can be determined by analyzing the virus released by a relatively small number of 3ZP-labeled infected cells (1 X 107). The validity of this method is demonstrated by the fact that cells labeled with 32P, as t,he precursor of phospholipid phosphorus, reach a steady state in the turnover of membrane phospholipid (Figs. 1 and 2). During this steady state the specific activity of the lipid phosphorus is the same for each of the individual phospholipid species, and therefore the distribution of the radioactivity in each species of phospholipids represents the actual distribution of the given phospholipid in the membrane (Fig. 3). Since the cells are growing and increasing in mass, and if the inorganic phosphate in the medium is the sole source of phosphorus for cellular components, the specific radioactivity of the total cellular phosphorus,
RNA
PHOSPHOLIPID
OF RNA KMOR
619
VIRGSF,s
and of each of the phosphorus-containing molecules, must asymptotically approach the specific radioactivity of the sole phosphorus precursor, provided the value of the latter is kept constant. The data presented here show that this expectation is fulfilled. ACKNOWLEDGMEiYTS The authors gratefully acknowledge the skilled technical assistance Janice M. Herbert and Miss Linda Steiner. J.P.Q. is a Leukemia Society of America Fellow. D.B.R.. is a Jane Coffin Childs Memorial Fund Fellow. The work was supported by a grant from the American Cancer Society
of
Miss
E-478.
REFERESCES 1. 2. 3. 4. 5. 6. 7.
QUIGLEY. J. P., RIFKIN, D. B., AND REICH, E.. TEMIN, H. M., Int. J. Cancer 3, 273 (1968). FOLCH, J., LEES, M.. AND SLOAN-STASLEY, J. SKIPSKI, V. P., PETERSON, R.. F., ASD BARCLAY, DITTMER, J. ASD LESTER, R., J. Lipid. Res. 5, BARTLETT, P., J. Biol. Chem. 234, 466 (1959). BRAY, G. A., Anal. Biochem. 1, 279 (1960).
Virology
46, 106 (1971).
B., J. Viol. Chem. 226, 497 (195 M., Biochem. J. 90, 374 (1964). 126 (1964).
‘I.