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Sialic acid in the cellular membranes of Ehrlich ascites-carcinoma cells
594
PRELIMINARY NOTES
Sialic acid in the cellular membranes of Ehrlich ascites-carclnoma cells Recent studies have demonstrated that sialic acid is present on the surfaces of erythrocytes from various animals and that it makes a major contribution to the negative surface charge of these cells z. The present investigation was designed to determine whether the negative electrophoretic mobility of Ehrlich ascites-carcinoma cells 2 was due to the presence of sialic acid on the cell surface, and whether sialic acid would appear in a specific subcellular fraction. The hyperdiploid Ehrlich ascites carcinoma was carried by weekly transfers in Swiss white mice. Tumors 5-8 days old and free of erythrocytes were used. IO ml of isotonic was injected into the peritoneal cavity of the host, the cell suspension withdrawn and the cells washed 3 times in 2o vol. of isotonic saline prior to suspension in a Krebs-Ringer phosphate medium. This washing procedure was found to be adequate to remove sialic acid contributed by the proteins of the ascites fluid. The proportion of cells in each cell suspension was determined by measuring the packed cell volume by centrifugation in a Wintrobe hematocrit tube at 45oo × g for 45 min. As determined previously 3 i ml of packed cells is equivalent to lO9 cells and to lO6 mg of dry weight. Sialic acid was measured by the method of WARREN4, after hydrolysis either by o.I N H2SO 4 for I h at 8o ° or by 5° units/ml of Cholera vibrio neuraminidase 5 at 37 ° for I h. Acid hydrolysis led to the release of a large amount of material producing a chromophore with an absorption maximum at 532 m/x, which could not be effectively removed by chromatography on Dowex-I or by extraction with isoamyl alcohol. Therefore, the absorbaney at 562 m/x due to neuraminic acid was corrected for absorbancy at 532 m/~ as proposed by WARREN4. N-acetyl neuraminic acid was used as a reference standard 6. The cells were ruptured by the pressure homogenization method of HUNTER AND COMMERFORD7 and separated into the various subcellular fractions as described previously by WALLACH3. Prior to the determination of sialic acid, sucrose-containing samples were extensively dialyzed against water since sucrose was found to interfere with the determination. The electrophoretic measurements were made at 25 ° in a micro-electrophoresis apparatus having a lateral cell and Ag, AgC1 electrodes. The buffer contained o.2ol M sucrose, o.oo59 M Na2HPO 4, O.OLO8M NaH~PO4 and 0.0441 M NaC1. The pH was 6. 4 and the ionic strength 0.o72. 16 determinations of the total sialic acid content of Ehrlich ascites-carcinoma cells were carried out, 12 using acid hydrolysis, and 4 enzymic hydrolysis of cell homogenates. The total sialic acid content, as determined by acid hydrolysis, was found to be 0.45 ± 0.08/xmole/ml of cells. The enzymic procedure gave a value of 0.42 =L o.oi/~mole/ml of cells. After removal of sialic acid associated with the ascitesfluid proteins, no further sialic acid could be removed by extensive washing of the cells in cold saline. When intact Ehrlich ascites-carcinoma cells (IOO mg/ml) were treated with Cholera vibrio neuraminidase (5° units/ml), 0.32 ± 0.o2 ~mole per ml of cells of sialic acid were removed. Although this represents 73 % of the total enzyme-hydrolyzable bound sialic acid, neuraminidase treatment produces no change in the respiration or glycolysis of these cells. Since the enzyme is too large e to penetrate into intact cells, these data suggest that most of the sialic acid is associated with the cell surface. Further support for this contention comes from the electrophoresis data. These I3iochim. Biophys..4cta, 52 (196I) 594-596
595
PRELIMINARY NOTES
showed that at p H 6.4 and ionic strength o.o72 Ehrlich ascites-carcinoma cells had a mobility of - - 1 2 . 1 . lO -5 cm*/V/sec corrected to the viscosity of water at 25 °. After treatment with neuraminidase, the mobility of the tumor cells was reduced by 72 % to - - 3 . 4 cm~/V/sec. The charge density a, was calculated from the mobilities, assuming a spherical shape and an infinite radius of curvature, from the relationship = - -
(1)
(ilk + a~)
where u is the electrophoretic mobility, ~ the viscosity, k is o.327.1o8.~v/I, I being the ionic strength, and a~ the radius of the counterion (Na +) taken as e.67 A. a was found to be 259o esu/cm ~ in untreated cells and 725 esu/cm ~ after neuraminidase treatment. Since these cells have a surface area of about 95o tz2, the difference, 1865 esu/cm 2, corresponds to about 37" Io6 negative charges/cell. However, assuming a molecular weight of 31o for N-acetyl neuraminic acid, the sialic acid removed from the intact cells corresponds to about 2o3 • lO6 molecules/cell, five times more than the change of charge. One possible explanation for this discrepancy is that in these cells the surface and ergastoplasmic membranes may be in dynamic continuity as proposed b y BENNETT8. It could then be that only one-fifth of the membrane-associated sialic acid is at the cell surface at any one instant, accounting for the 37" zoe negative charges per cell contributed by this material. Over a period of time, however, the continuous interchange between surface and ergastoplasmic membranes would present far more sialic acid to the action of the neuraminidase than would affect the surface charge. TABLE I SIALIC
ACID
Fraction
IN
SUBCELLULAR
FRACTIONS
Sialic acid* (l~mole/mlcells)
%
Cells
o.382
i oo
Nuclei Mitochondria Microsomes Soluble
o.oi2 o.o66 o.265 0.052
3.z 17.3 69.5 13.6
Sum
0.395
lO3.6
Some support for this concept arises from the distribution of sialic acid in the various subcellular fractions. As shown in Table I, about two-thirds of the total sialic acid is found in the microsome fraction. 98 % of this is removed if the cells are exposed to neuraminidase prior to rupture. Neuraminidase treatment also removes one-third to one-half of the sialic acid associated with the nuclei, mitochondria and soluble fractions. When the microsomes are further fractionated b y centrifugation in sucrose gradients between 1.151 and 1.241 specific gravity, at lO5 ooo x g for 16 h, 76 % of the sialic acid is found in an RNA-free membrane fraction floating at specific gravity i. 18, and the remainder in an RNA-free membrane fraction floating at specific gravity 1.22. Biochim. Biophys. Acta, 52 (1961) 594-596
596
PRELIMINARY NOTES
Both the effect of neuraminidase on the intact cell and the distribution of sialic acid in the subcellular fractions are consistent with the concept of membrane translation. One would, however, expect such a process to be depressed by metabolic inhibitors, yet poisoning of the cells with 2,4 dinitrophenol (i0 -4 M) or CN- (I0 3 M) prior to, or coincident with, exposure to neuraminidase, produced no effect upon sialic acid removal by the enzyme, even in the absence of glucose. The discrepancy between the change of charge and amount of sialic acid removed by enzyme action may equally be explained by the suggestion that here, as well as in the case of erythrocytes z, assumption of an infinite radius of curvature underestimates the true surface charge. If, as has been suggested by BR1NTONAND LAUFFER9 for human erythrocytes, there is a finite, 2o-4o-,A, radius of curvature at the sialic acid loci, there would be a far better correlation between sialic acid removed and change in surface charge. If this is the case, the distribution data would indicate that, while there is some sialic acid in all cellular membranes, most of it is on the surface membrane, which sediments with the microsomes after cell rupture. This work has been supported by grants from the U.S. Public Health Service.
Department of Biological Chemistry, Harvard Medical School, and Protein Foundation, Boston, Mass. (U.S.A.)
DONALD F. HOELZL WALLACH E D W I N H . EYLAR
1 E. H. EYLAR, M. A. MADOFF, O. V. BRODY AND J. L. ONCLEY, to be published. 2 L. PURDOM, E. J. AMBROSE AND G. KLEIN, Nature, 181 (1958) I586. 3 D. F. H. WALLACH, J. SODERBERG AND L. BRICRER, Cancer Research, 20 (196o) 3974 L. WARREN, J. Biol. Chem., 234 (1959) 1971. 5 M. A. MADOEF, E. H. EYLAR AND L. WE1NSTEIN, J. [mmunol., 8.5 (2960) 603. 6 E. H. EYLAR, P h . D . Thesis, Harvard University, i958. 7 M. J. HUNTER AND S. L. COMMERFORD, Biochim. Biophys. Acta, 47 (1961) 580. 8 H. S. BENNETT, jr. Biophys. Biochem. Cytol., 2 (I956) 90. 9 C. C. BRINTON, Jr. AND M. A. LAUFFER, in M. BIER, Electrophoresis, Theory, Methods and Applications, Academic Press, Inc., New Y~rk, 1959, p. 427 •
Received August I I t h , I96I Biochim. Biopl~ys. Acta, 52 (t961) 594 596
The effect of parathyroid hormone on the oxidation of carbohydrate by bone It has been reported that parathyroid hormone causes demineralization of osseous tissue by inducing in bone an accumulation of citrate which then chelates Ca 2+ (refs. 1-3). Since the metabolic alteration responsible for this accumulation has not been illucidated, we reinvestigated this problem as part of a larger study on the metabolism of osseous tissue. 75o-iooo units of parathyroid extract (Eli Lilly) were administered in a single subcutaneous injection to weanling rabbits at time schedules shown in Table I. At the time of sacrifice, the serum Ca of all of the treated animals had increased from Biochim. Biophys. Acta, 52 (1961) 596 599
PRELIMINARY NOTES
Sialic acid in the cellular membranes of Ehrlich ascites-carclnoma cells Recent studies have demonstrated that sialic acid is present on the surfaces of erythrocytes from various animals and that it makes a major contribution to the negative surface charge of these cells z. The present investigation was designed to determine whether the negative electrophoretic mobility of Ehrlich ascites-carcinoma cells 2 was due to the presence of sialic acid on the cell surface, and whether sialic acid would appear in a specific subcellular fraction. The hyperdiploid Ehrlich ascites carcinoma was carried by weekly transfers in Swiss white mice. Tumors 5-8 days old and free of erythrocytes were used. IO ml of isotonic was injected into the peritoneal cavity of the host, the cell suspension withdrawn and the cells washed 3 times in 2o vol. of isotonic saline prior to suspension in a Krebs-Ringer phosphate medium. This washing procedure was found to be adequate to remove sialic acid contributed by the proteins of the ascites fluid. The proportion of cells in each cell suspension was determined by measuring the packed cell volume by centrifugation in a Wintrobe hematocrit tube at 45oo × g for 45 min. As determined previously 3 i ml of packed cells is equivalent to lO9 cells and to lO6 mg of dry weight. Sialic acid was measured by the method of WARREN4, after hydrolysis either by o.I N H2SO 4 for I h at 8o ° or by 5° units/ml of Cholera vibrio neuraminidase 5 at 37 ° for I h. Acid hydrolysis led to the release of a large amount of material producing a chromophore with an absorption maximum at 532 m/x, which could not be effectively removed by chromatography on Dowex-I or by extraction with isoamyl alcohol. Therefore, the absorbaney at 562 m/x due to neuraminic acid was corrected for absorbancy at 532 m/~ as proposed by WARREN4. N-acetyl neuraminic acid was used as a reference standard 6. The cells were ruptured by the pressure homogenization method of HUNTER AND COMMERFORD7 and separated into the various subcellular fractions as described previously by WALLACH3. Prior to the determination of sialic acid, sucrose-containing samples were extensively dialyzed against water since sucrose was found to interfere with the determination. The electrophoretic measurements were made at 25 ° in a micro-electrophoresis apparatus having a lateral cell and Ag, AgC1 electrodes. The buffer contained o.2ol M sucrose, o.oo59 M Na2HPO 4, O.OLO8M NaH~PO4 and 0.0441 M NaC1. The pH was 6. 4 and the ionic strength 0.o72. 16 determinations of the total sialic acid content of Ehrlich ascites-carcinoma cells were carried out, 12 using acid hydrolysis, and 4 enzymic hydrolysis of cell homogenates. The total sialic acid content, as determined by acid hydrolysis, was found to be 0.45 ± 0.08/xmole/ml of cells. The enzymic procedure gave a value of 0.42 =L o.oi/~mole/ml of cells. After removal of sialic acid associated with the ascitesfluid proteins, no further sialic acid could be removed by extensive washing of the cells in cold saline. When intact Ehrlich ascites-carcinoma cells (IOO mg/ml) were treated with Cholera vibrio neuraminidase (5° units/ml), 0.32 ± 0.o2 ~mole per ml of cells of sialic acid were removed. Although this represents 73 % of the total enzyme-hydrolyzable bound sialic acid, neuraminidase treatment produces no change in the respiration or glycolysis of these cells. Since the enzyme is too large e to penetrate into intact cells, these data suggest that most of the sialic acid is associated with the cell surface. Further support for this contention comes from the electrophoresis data. These I3iochim. Biophys..4cta, 52 (196I) 594-596
595
PRELIMINARY NOTES
showed that at p H 6.4 and ionic strength o.o72 Ehrlich ascites-carcinoma cells had a mobility of - - 1 2 . 1 . lO -5 cm*/V/sec corrected to the viscosity of water at 25 °. After treatment with neuraminidase, the mobility of the tumor cells was reduced by 72 % to - - 3 . 4 cm~/V/sec. The charge density a, was calculated from the mobilities, assuming a spherical shape and an infinite radius of curvature, from the relationship = - -
(1)
(ilk + a~)
where u is the electrophoretic mobility, ~ the viscosity, k is o.327.1o8.~v/I, I being the ionic strength, and a~ the radius of the counterion (Na +) taken as e.67 A. a was found to be 259o esu/cm ~ in untreated cells and 725 esu/cm ~ after neuraminidase treatment. Since these cells have a surface area of about 95o tz2, the difference, 1865 esu/cm 2, corresponds to about 37" Io6 negative charges/cell. However, assuming a molecular weight of 31o for N-acetyl neuraminic acid, the sialic acid removed from the intact cells corresponds to about 2o3 • lO6 molecules/cell, five times more than the change of charge. One possible explanation for this discrepancy is that in these cells the surface and ergastoplasmic membranes may be in dynamic continuity as proposed b y BENNETT8. It could then be that only one-fifth of the membrane-associated sialic acid is at the cell surface at any one instant, accounting for the 37" zoe negative charges per cell contributed by this material. Over a period of time, however, the continuous interchange between surface and ergastoplasmic membranes would present far more sialic acid to the action of the neuraminidase than would affect the surface charge. TABLE I SIALIC
ACID
Fraction
IN
SUBCELLULAR
FRACTIONS
Sialic acid* (l~mole/mlcells)
%
Cells
o.382
i oo
Nuclei Mitochondria Microsomes Soluble
o.oi2 o.o66 o.265 0.052
3.z 17.3 69.5 13.6
Sum
0.395
lO3.6
Some support for this concept arises from the distribution of sialic acid in the various subcellular fractions. As shown in Table I, about two-thirds of the total sialic acid is found in the microsome fraction. 98 % of this is removed if the cells are exposed to neuraminidase prior to rupture. Neuraminidase treatment also removes one-third to one-half of the sialic acid associated with the nuclei, mitochondria and soluble fractions. When the microsomes are further fractionated b y centrifugation in sucrose gradients between 1.151 and 1.241 specific gravity, at lO5 ooo x g for 16 h, 76 % of the sialic acid is found in an RNA-free membrane fraction floating at specific gravity i. 18, and the remainder in an RNA-free membrane fraction floating at specific gravity 1.22. Biochim. Biophys. Acta, 52 (1961) 594-596
596
PRELIMINARY NOTES
Both the effect of neuraminidase on the intact cell and the distribution of sialic acid in the subcellular fractions are consistent with the concept of membrane translation. One would, however, expect such a process to be depressed by metabolic inhibitors, yet poisoning of the cells with 2,4 dinitrophenol (i0 -4 M) or CN- (I0 3 M) prior to, or coincident with, exposure to neuraminidase, produced no effect upon sialic acid removal by the enzyme, even in the absence of glucose. The discrepancy between the change of charge and amount of sialic acid removed by enzyme action may equally be explained by the suggestion that here, as well as in the case of erythrocytes z, assumption of an infinite radius of curvature underestimates the true surface charge. If, as has been suggested by BR1NTONAND LAUFFER9 for human erythrocytes, there is a finite, 2o-4o-,A, radius of curvature at the sialic acid loci, there would be a far better correlation between sialic acid removed and change in surface charge. If this is the case, the distribution data would indicate that, while there is some sialic acid in all cellular membranes, most of it is on the surface membrane, which sediments with the microsomes after cell rupture. This work has been supported by grants from the U.S. Public Health Service.
Department of Biological Chemistry, Harvard Medical School, and Protein Foundation, Boston, Mass. (U.S.A.)
DONALD F. HOELZL WALLACH E D W I N H . EYLAR
1 E. H. EYLAR, M. A. MADOFF, O. V. BRODY AND J. L. ONCLEY, to be published. 2 L. PURDOM, E. J. AMBROSE AND G. KLEIN, Nature, 181 (1958) I586. 3 D. F. H. WALLACH, J. SODERBERG AND L. BRICRER, Cancer Research, 20 (196o) 3974 L. WARREN, J. Biol. Chem., 234 (1959) 1971. 5 M. A. MADOEF, E. H. EYLAR AND L. WE1NSTEIN, J. [mmunol., 8.5 (2960) 603. 6 E. H. EYLAR, P h . D . Thesis, Harvard University, i958. 7 M. J. HUNTER AND S. L. COMMERFORD, Biochim. Biophys. Acta, 47 (1961) 580. 8 H. S. BENNETT, jr. Biophys. Biochem. Cytol., 2 (I956) 90. 9 C. C. BRINTON, Jr. AND M. A. LAUFFER, in M. BIER, Electrophoresis, Theory, Methods and Applications, Academic Press, Inc., New Y~rk, 1959, p. 427 •
Received August I I t h , I96I Biochim. Biopl~ys. Acta, 52 (t961) 594 596
The effect of parathyroid hormone on the oxidation of carbohydrate by bone It has been reported that parathyroid hormone causes demineralization of osseous tissue by inducing in bone an accumulation of citrate which then chelates Ca 2+ (refs. 1-3). Since the metabolic alteration responsible for this accumulation has not been illucidated, we reinvestigated this problem as part of a larger study on the metabolism of osseous tissue. 75o-iooo units of parathyroid extract (Eli Lilly) were administered in a single subcutaneous injection to weanling rabbits at time schedules shown in Table I. At the time of sacrifice, the serum Ca of all of the treated animals had increased from Biochim. Biophys. Acta, 52 (1961) 596 599