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Analysis of myelin-synaptosomal interactions in sucrose and ficoll-sucrose gradients
ANALYTICAL
Analysis
BIOCHEMISTRY
49,
%36
(1972)
of Myelin-Synaptosomal and
Ficoll-Sucrose
L. C. DOYLE Department
Interactions
of Psychobiology, Irvine,
AND
in Sucrose
Gradients c.
w.
COTMAN
University of Calijornia-Irvine, California 92664
Received October 28,
1971
A critical assumption underlying all strategies of subcellular separations is the absence of particle-particle int.eractions. The complete resolution of subcellular particles by centrifugation procedures requires that particles sediment independently. Otherwise, particle aggregates will cosediment, prohibiting the separation of individual particles. When aggregrated particles are isopycnically banded, they will equilibrate together without separating into components ; and, during rate sedimentation, particle aggregrates lead to anomalous sedimentation behavior. Recently, this assumption that particles do not interact with each other has been questioned by Day and coworkers (1) for subcellular separations in brain. Day and coworkers observed differences in buoyant densities for classes of brain subcellular particles in Ficoll-sucrose versus sucrose gradients. They attributed this to an interaction between particles such as synaptosomes and myelin in sucrose gradients, but not in Ficollsucrose gradients. A reasonable alternative to their hypothesis is that differences in density result from differences in osmotic propert’ies of these two media. On the basis of their data, there is no way to positively distinguish between these two hypotheses. Because of possible adverse effects on separations, it is essential to identify particle-particle interactions when they exist and the conditions under which they exist. In this paper, we examine the possibility that synaptosomes and myelin particles interact during sedimentation. We demonstrate that fractions of purified synaptosomes and purified myelin sediment essentially independently and can find no evidence for an interaction. The difference in buoyant densities they observed in Ficoll-sucrose vs sucrose gradients are due to the osmotic properties of different gradient media acting on individual particles. A way to assess particle-particle interactions is to measure the sedimentation behavior of each particle first independently, and then in a mixture of the two particles (2). If no interact,ions occur, both results 29
@
1972 by Academic Press, Inc. All rights of reproduction in any
form
reserved.
30 will be analyze purified ture of
DOYLE
AND
COTMAN
identical. To test whether myelin the isopycnic banding profiles myelin and purified synaptosomes both in sucrose and Ficoll-surcose
and synaptosomes interact, we and enzyme distributions for independently and for a mixgradients.
EXPERIMENTAL
Myelin was purified from rat forebrain by the method of Autilio, Norton, and Terry (3) adapted for the 30 rotor. Synaptosomes were purified from rat forebrain, as described by Cotman and Matthews (4). Discontinuous gradients of sucrose (w/w), Ficoll (w/v), and Ficoll (w/v) in 0.32 M sucrose were prepared with four density steps (1.06, 1.07, 1.12, 1.15 gm/cc) of 5 ml each. The density of Ficoll-sucrose solutions was measured by a gravitometer, while the densities of sucrose and Ficoll at 5°C were determined from tables relating densities and refractive indices provided by Pharmacia for Ficoll and equations derived by Barber (5) for sucrose. The refractive index was measured with the Abbe refractometer. 2 ml aliquots of purified fractions in 0.32 M sucrose (0.40.6 mg protein/ml) and a mixture of synaptosomes and myelin (1: 1 with respect to protein content) were layered onto the discontinuous gradients and centrifuged in an SW 25.1 rotor at 58,OOOg (R,,) for 3 hr. Gradient fractions were separated with a Beckman tube cutter, diluted in 0.32 M sucrose (at least 4 vol), pelleted at 106,OOOg (R,,,) for 30 min, and resuspended in a known volume of 0.32 M sucrose. Prior to assay, samples were freeze-thawed one to two times. Protein was measured by the method of Lowry (6). Alkaline phosphatase (EC 3.1.3.1) was measured in 0.05 M 2-amino-2-methyl-1-propanol, 2 mM CgC12, pH 10.0, with 3.8 mM p-nitrophenyl phosphate (7). Cytochrome oxidase activity (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was assayed by the method of Duncan and Mackler (8). The limit of detectability is 5 X 10e5 pmole reduced cytochrome c oxidized/min. RESULTS
AND DISCUSSION
The effect of the gradient medium on particle density is shown in Fig. 1. The equilibrium density of myelin alone or synaptosomes alone clearly depends on the centrifugation media. Myelin bands between 1.07 and 1.12 gm/cc on sucrose gradients (Fig. 1A). In discontinuous Ficoll-sucrose gradients, myelin bands at 1.06 and 1.07 gm/cc (Fig. 1B). Purified synaptosomes isolated from a Ficoll-sucrose gradient also have a lighter isopycnic banding density in Ficoll and Ficoll-sucrose gradients than in sucrose gradients. In sucrose gradients, the majority of the synaptosomes band at 1.15 gm/cc with a minor component at 1.12 gmJcc (below first bead in Fig. 2A). However, in Ficoll-sucrose gradients, the majority of the synaptosomes band at 1.12 gm/cc, with only a minor component
MTELIN-SYNAPTOSOMAL
INTERACTIONS
31
F ‘IG. 1. Isopycnic banding profiles of purified myelin in (A) sucrose (w/w) (w/v) in 0.32 M sucrose. The gradients were composed of four density (B) Ficoll at 1 .06, 1.07, 1.12, and 1.15 gm/cc, upon which 2 ml of purified myelin was la! Cen trifugation in SW 25.1 rotor at 58,OOOg for 3 hr brought the particles to isop ycnic banding densities (6 hr did not change the dktrihution!. The beads den sities of (1) 1.09 and (2) 1.15 gm/cc.
k’ru. 2. Isopycnic Ficoli-sucrose (B)
banding gradients.
profiles of purified synaptosomes in sucrose (A) :iud Experimental conditions are t.he same as for Fig. I.
32
DOYLE
AND
COTMAN
FIG. 3. Isopyenic banding profiles of purified synaptosomes mixed with an approximately equal amount of purified myelin. Gradients are (A) sucrose (W/W) and (B) Ficoll (w/v) in 0.32M sucrose. Experimental conditions are the same as for Fig. 1.
reaching 1.15 gm/cc. Gradients that contain both particle populations appear as an overlay of the two gradients that each contain only one of these particle populations (Fig. 3). Thus, our data agree with those of Day et al. (1) in that particle densities are lighter in Ficoll-sucrose than in sucrose gradients. However, since only one particle is present, the densities are not primarily due to interactions between myelin and synaptosomes. To test for myelin-synaptosomal interactions not observable in the isopycnic banding profiles, we analyzed the distribution of protein, alkaline phosphatase (a plasma membrane marker) (4), and cytochrome oxidase (a mitochondrial marker) in sucrose and Ficoll-sucrose gradients containing synaptosomes alone, myelin alone, or both mixed 1: 1 with respect to protein content. The distributions of protein and alkaline phosphatase in gradients with both synaptosomes and myelin do not differ dramatically from the expected if no interactions occurred. Protein distributions in sucrose gradients reveal that 3% of the total protein has been added to the upper fraction and 3% has been removed from the lower fraction. For protein distributions in Ficoll-sucrose gradients, the deviation from expected is -3% for the upper fraction and +3% for the lower fraction. For alkaline phosphatase distributions in sucrose gradients, 2% of the total alkaline phosphatase is lost from the lower gradient
MYELIN-SYNAPTOSOMAL
INTERACTIONS
TABLE 1 Protein Distribution in Sucrose and Ficoll-Sucrose C;radienB Containing Synaptosomes Alone, Myelin Alone, and the Actual and Expected Combination of Both Particles Mixed 1: 1 Gradients were divided into two fractions: above 1.12 gm,/cc (A) and below 1.12 gm/cc (B). Deviation from expected for a gradient fraction, for example gradient fraction A, is computed by the difference between the actual per cent of total (A,/(A, + B,)) and the expected per cent of total (AJ(AB + B,)). Protein, mg,!gradient (iradient. fraction Sucrose: A 13
b J naptosomes sr alone (+)
frac.tion or sample zone ~~~ _ ~~~ ,2lgelill AVtll:ll I’:xpect ed alone ($) combination combination
0.016 0. 76.5
U.513 0 0 I :;
0.5h2 0.77::
0 529 0 77s
To (.a1 Recovery (ii ) Ficoll-srrrrose: A 13
(54)
1 XL5 t 59 )
1 ;;o‘i
(63) 0.042 0.707
0,406 0.070
0.516 0 s57
0 3:zs 0.77’7
Total Recovery- (‘;;,) Sample zone
(5S) 0. !I80
1 :;‘i:; (60) 2.2s;;
1 :; I 5
(62) 1.210
2. 190
I)eviatiorl (‘,; total),‘ +3 -r>.,
-3 $3
+4
* The results of this experiment are similar (within lOTo) t,o those from t,wo other experiment’s in which protein distributions were analyzed.
fraction and 2% is gained in the upper gradient fraction. In Ficoll-sucrose gradients, 2% of total alkaline phosphatase is lost from the lower fraction and 25%gained by the upper. Therefore, less than 3% of the protein or alkaline phosphatase activity shifts to a significantly different isopycnic banding density due to myelin-synaptosomal interactions. Cytochrome oxidase, which represents primarily intrasynaptosomal mitochondria, does not change its distribution at all when synaptosomes and myelin are intermixed. Cytochrome oxidase bands in the lower fraction of the gradient with synaptosomes alone and does not appear in the gradient containing myelin alone (Table 3). When synaptosomes and myelin are mixed, 100% of the cytochrome oxidase activity appears in the lower fraction of the gradient. Thus, there is no evidence of myelinsynaptosomal interactions assayed by cytochrome oxidase activity. Our data show that the difference in equilibrium densities for myelin and synaptosomes in sucrose versus Ficoll-sucrose gradients are due to differences in particle densities in sucrose versus Ficoll-sucrose gradients
34
DOYLE
AND
COTMAN
TABLE
2
Alkaline Phosphatase Distribution in Sucrose and Ficoll-Sucrose Gradients Containing Synaptosomes Alone, Myelm Alone, and the Actual and Expected Combination of Both Particles Mixed 1: 1 The deviation gradient fraction (A./(A. + B,)) divided into two
of the actual from the expected for a gradient fraction, for example A, is computed by the difference between the actual per cent of total and the expected per cent of total (LB/(& + B,)). Gradients were fractions, above 1.12 gm/cc (A) and below 1.12 gm/cc (B). Pi formed, mNmoles/min/gradient fraction or sample zone
Gradient fraction Sucrose : A B Total Recovery (%I Ficoll-sucrose: A B Total ltecovery (ye) Sample zone
Synaptosomes alone (i )
Myelin alone (3)
Actual combination
Expected combination
Deviation ( yO total)a
-2
0.17 7.14
0.15 0.09
0.42 6.48
0.32 7.23
(48)
6.90 (77)
7.55
(851
0.84 6.84
0.49
0.35
1.00
6.71
0. 13
6.46
(84) 8.58
(96) 0.50
7.46 (84) 8.92
0 These results are representative (within 10yo) alkaline phosphatase distributions were analyzed.
of another
+2
i-2 -2
7.68 9.08
experiment
-2
in which
rather than particle-particle interactions. Firmly established principles explain these differences in particle densities. The buoyant density depends in part on the permeability of the particles to the gradient solute (9,lO). Ficoll-sucrose gradients are essentially isosmotic, and thus do not cause osmotic dehydration which would result in an increase in particle density. Sucrose gradients in the range of concentrations used for isopycnic banding of subcellular particles are hypertonic, and thus promote the dehydration of the particles which makes the particles more dense. As a result, particles with an intravesicular compartment permeable to sucrose, but not to Ficoll, are less dense in Ficoll-sucrose gradients than in sucrose gradients. Day and coworkers (1) also interpret their data based on rate zonal sedimentation in discontinuous sucrose gradients as evidence for particleparticle interactions. We find these data difficult to interpret since measurement of sedimentation changes by light-scattering profiles will
MYELIN-SYNAPTOSOMAL
35
INTERACTIONS
TABLE 3 Cytochrome Oxidase Distribution in Sucrose and Ficoll-Sucrose Gradients Cont’aining Myelin Alone, Synaptosomes Alone, and the Actual and Expected Combination of Both (1: 1) Gradients gdcc
were divided into two fractions:
above 1.12 gm/cc (A) and below
1.12
(B).
Per cent of total cyt,ochrome oxidane Gradient fraction Sucrose : A B Ficoll-sucrose : A B
Myelin alone (4)
Synaptosomes alone ($1
Sctual combinat,ion
0.0 0.0
100.0
100.
0.0 0.0
0. 0 100 0
0.0 100.0
0. 0
Expected combination
0.0 0
0.0 100.0
0. 0 100.0
The data represent results from three experiments. lOOv/, of cytochrome oxidase represents 138 to 318 X 10m4pmole cytochrome c oxidized/min/gradient fraction, where the limit of detectability is 0.5 X 10m4pmole cytochrome c oxidized/min.
not reveal the discrete sedimentation of particular particle populations. We have ourselves analyzed the rate sedimentation of mitochondria and synaptosomes from brain homogenates in a variety of sucrose gradients. Our results could be quantitatively described by the Svedberg equation, which assumes that no particle-particle interactions occur (11). Thus, we find no evidence for myelin-synaptosomal interactions based on analysis of isopycnic or rate zonal centrifugation and conclude that, if such interactions do exist, they must be minimal. SCKNOWLEDGMENT This research was supported and Stroke (NS 08597).
by the National
Institute
of Neurological
Diseases
REFERENCES 1.
E. D.. MCMILLAN, P. N., MICKEY, D. D.. AND APPEL, S. H., Ad. Rioch,em. 39, 29 (1971). 2. MARTIN, R. G., AND AMES, B. N., J. Biol. Chem. 236, 1372 (1961). 3. AUTILIO. I,. A., NORTON, W. T., AND TERRY, R. D., J. Neurochem. 11, 17 (1964). 4. COTMAN, C. W., AND MATTHEWS, D. A., Biochim. Biophys. Acta 249, 380 (1971). 5. EIRBER. E. J., Nut. Cancer Isst. Mono~rq~h 21, 219 (1966). 6. LOWRY, 0. H., ROSEBROUGH. N. J., F~RR, A. I,., .WD RANI)ALL. R. J., J. Biol. Chem. 193, 265 (1951). 7. LOWRY, 0. H., ROBERTS, N. R., WIR. M., HIXON, W. S.. AND CRAMTFORD, E. J., J. Riol. CILem. 207, 19 (1954). DAY.
36
DOYLE
AND
COTMAN
8. DUNCAN, H. M., AND MACKLER, B. J., J. Biol. Chem. 241, 1694 (1966). 9. STECK, T. L., STRAUS, J. H., AND WALLACH, D. F. H., Biochim. Biohpys. Acta 203, 385 (1970). 10. WALLACH, D. F. H., in “The Specificity of Cell Surfaces” (B. D. Davis and L. Warren, eds.), p. 129. Prentice-Hall, Englewood Cliffs, 1967. 11. COTMAN, C., AND DOYLE, L., Arch. Biochem. Biophys., in press.
Analysis
BIOCHEMISTRY
49,
%36
(1972)
of Myelin-Synaptosomal and
Ficoll-Sucrose
L. C. DOYLE Department
Interactions
of Psychobiology, Irvine,
AND
in Sucrose
Gradients c.
w.
COTMAN
University of Calijornia-Irvine, California 92664
Received October 28,
1971
A critical assumption underlying all strategies of subcellular separations is the absence of particle-particle int.eractions. The complete resolution of subcellular particles by centrifugation procedures requires that particles sediment independently. Otherwise, particle aggregates will cosediment, prohibiting the separation of individual particles. When aggregrated particles are isopycnically banded, they will equilibrate together without separating into components ; and, during rate sedimentation, particle aggregrates lead to anomalous sedimentation behavior. Recently, this assumption that particles do not interact with each other has been questioned by Day and coworkers (1) for subcellular separations in brain. Day and coworkers observed differences in buoyant densities for classes of brain subcellular particles in Ficoll-sucrose versus sucrose gradients. They attributed this to an interaction between particles such as synaptosomes and myelin in sucrose gradients, but not in Ficollsucrose gradients. A reasonable alternative to their hypothesis is that differences in density result from differences in osmotic propert’ies of these two media. On the basis of their data, there is no way to positively distinguish between these two hypotheses. Because of possible adverse effects on separations, it is essential to identify particle-particle interactions when they exist and the conditions under which they exist. In this paper, we examine the possibility that synaptosomes and myelin particles interact during sedimentation. We demonstrate that fractions of purified synaptosomes and purified myelin sediment essentially independently and can find no evidence for an interaction. The difference in buoyant densities they observed in Ficoll-sucrose vs sucrose gradients are due to the osmotic properties of different gradient media acting on individual particles. A way to assess particle-particle interactions is to measure the sedimentation behavior of each particle first independently, and then in a mixture of the two particles (2). If no interact,ions occur, both results 29
@
1972 by Academic Press, Inc. All rights of reproduction in any
form
reserved.
30 will be analyze purified ture of
DOYLE
AND
COTMAN
identical. To test whether myelin the isopycnic banding profiles myelin and purified synaptosomes both in sucrose and Ficoll-surcose
and synaptosomes interact, we and enzyme distributions for independently and for a mixgradients.
EXPERIMENTAL
Myelin was purified from rat forebrain by the method of Autilio, Norton, and Terry (3) adapted for the 30 rotor. Synaptosomes were purified from rat forebrain, as described by Cotman and Matthews (4). Discontinuous gradients of sucrose (w/w), Ficoll (w/v), and Ficoll (w/v) in 0.32 M sucrose were prepared with four density steps (1.06, 1.07, 1.12, 1.15 gm/cc) of 5 ml each. The density of Ficoll-sucrose solutions was measured by a gravitometer, while the densities of sucrose and Ficoll at 5°C were determined from tables relating densities and refractive indices provided by Pharmacia for Ficoll and equations derived by Barber (5) for sucrose. The refractive index was measured with the Abbe refractometer. 2 ml aliquots of purified fractions in 0.32 M sucrose (0.40.6 mg protein/ml) and a mixture of synaptosomes and myelin (1: 1 with respect to protein content) were layered onto the discontinuous gradients and centrifuged in an SW 25.1 rotor at 58,OOOg (R,,) for 3 hr. Gradient fractions were separated with a Beckman tube cutter, diluted in 0.32 M sucrose (at least 4 vol), pelleted at 106,OOOg (R,,,) for 30 min, and resuspended in a known volume of 0.32 M sucrose. Prior to assay, samples were freeze-thawed one to two times. Protein was measured by the method of Lowry (6). Alkaline phosphatase (EC 3.1.3.1) was measured in 0.05 M 2-amino-2-methyl-1-propanol, 2 mM CgC12, pH 10.0, with 3.8 mM p-nitrophenyl phosphate (7). Cytochrome oxidase activity (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was assayed by the method of Duncan and Mackler (8). The limit of detectability is 5 X 10e5 pmole reduced cytochrome c oxidized/min. RESULTS
AND DISCUSSION
The effect of the gradient medium on particle density is shown in Fig. 1. The equilibrium density of myelin alone or synaptosomes alone clearly depends on the centrifugation media. Myelin bands between 1.07 and 1.12 gm/cc on sucrose gradients (Fig. 1A). In discontinuous Ficoll-sucrose gradients, myelin bands at 1.06 and 1.07 gm/cc (Fig. 1B). Purified synaptosomes isolated from a Ficoll-sucrose gradient also have a lighter isopycnic banding density in Ficoll and Ficoll-sucrose gradients than in sucrose gradients. In sucrose gradients, the majority of the synaptosomes band at 1.15 gm/cc with a minor component at 1.12 gmJcc (below first bead in Fig. 2A). However, in Ficoll-sucrose gradients, the majority of the synaptosomes band at 1.12 gm/cc, with only a minor component
MTELIN-SYNAPTOSOMAL
INTERACTIONS
31
F ‘IG. 1. Isopycnic banding profiles of purified myelin in (A) sucrose (w/w) (w/v) in 0.32 M sucrose. The gradients were composed of four density (B) Ficoll at 1 .06, 1.07, 1.12, and 1.15 gm/cc, upon which 2 ml of purified myelin was la! Cen trifugation in SW 25.1 rotor at 58,OOOg for 3 hr brought the particles to isop ycnic banding densities (6 hr did not change the dktrihution!. The beads den sities of (1) 1.09 and (2) 1.15 gm/cc.
k’ru. 2. Isopycnic Ficoli-sucrose (B)
banding gradients.
profiles of purified synaptosomes in sucrose (A) :iud Experimental conditions are t.he same as for Fig. I.
32
DOYLE
AND
COTMAN
FIG. 3. Isopyenic banding profiles of purified synaptosomes mixed with an approximately equal amount of purified myelin. Gradients are (A) sucrose (W/W) and (B) Ficoll (w/v) in 0.32M sucrose. Experimental conditions are the same as for Fig. 1.
reaching 1.15 gm/cc. Gradients that contain both particle populations appear as an overlay of the two gradients that each contain only one of these particle populations (Fig. 3). Thus, our data agree with those of Day et al. (1) in that particle densities are lighter in Ficoll-sucrose than in sucrose gradients. However, since only one particle is present, the densities are not primarily due to interactions between myelin and synaptosomes. To test for myelin-synaptosomal interactions not observable in the isopycnic banding profiles, we analyzed the distribution of protein, alkaline phosphatase (a plasma membrane marker) (4), and cytochrome oxidase (a mitochondrial marker) in sucrose and Ficoll-sucrose gradients containing synaptosomes alone, myelin alone, or both mixed 1: 1 with respect to protein content. The distributions of protein and alkaline phosphatase in gradients with both synaptosomes and myelin do not differ dramatically from the expected if no interactions occurred. Protein distributions in sucrose gradients reveal that 3% of the total protein has been added to the upper fraction and 3% has been removed from the lower fraction. For protein distributions in Ficoll-sucrose gradients, the deviation from expected is -3% for the upper fraction and +3% for the lower fraction. For alkaline phosphatase distributions in sucrose gradients, 2% of the total alkaline phosphatase is lost from the lower gradient
MYELIN-SYNAPTOSOMAL
INTERACTIONS
TABLE 1 Protein Distribution in Sucrose and Ficoll-Sucrose C;radienB Containing Synaptosomes Alone, Myelin Alone, and the Actual and Expected Combination of Both Particles Mixed 1: 1 Gradients were divided into two fractions: above 1.12 gm,/cc (A) and below 1.12 gm/cc (B). Deviation from expected for a gradient fraction, for example gradient fraction A, is computed by the difference between the actual per cent of total (A,/(A, + B,)) and the expected per cent of total (AJ(AB + B,)). Protein, mg,!gradient (iradient. fraction Sucrose: A 13
b J naptosomes sr alone (+)
frac.tion or sample zone ~~~ _ ~~~ ,2lgelill AVtll:ll I’:xpect ed alone ($) combination combination
0.016 0. 76.5
U.513 0 0 I :;
0.5h2 0.77::
0 529 0 77s
To (.a1 Recovery (ii ) Ficoll-srrrrose: A 13
(54)
1 XL5 t 59 )
1 ;;o‘i
(63) 0.042 0.707
0,406 0.070
0.516 0 s57
0 3:zs 0.77’7
Total Recovery- (‘;;,) Sample zone
(5S) 0. !I80
1 :;‘i:; (60) 2.2s;;
1 :; I 5
(62) 1.210
2. 190
I)eviatiorl (‘,; total),‘ +3 -r>.,
-3 $3
+4
* The results of this experiment are similar (within lOTo) t,o those from t,wo other experiment’s in which protein distributions were analyzed.
fraction and 2% is gained in the upper gradient fraction. In Ficoll-sucrose gradients, 2% of total alkaline phosphatase is lost from the lower fraction and 25%gained by the upper. Therefore, less than 3% of the protein or alkaline phosphatase activity shifts to a significantly different isopycnic banding density due to myelin-synaptosomal interactions. Cytochrome oxidase, which represents primarily intrasynaptosomal mitochondria, does not change its distribution at all when synaptosomes and myelin are intermixed. Cytochrome oxidase bands in the lower fraction of the gradient with synaptosomes alone and does not appear in the gradient containing myelin alone (Table 3). When synaptosomes and myelin are mixed, 100% of the cytochrome oxidase activity appears in the lower fraction of the gradient. Thus, there is no evidence of myelinsynaptosomal interactions assayed by cytochrome oxidase activity. Our data show that the difference in equilibrium densities for myelin and synaptosomes in sucrose versus Ficoll-sucrose gradients are due to differences in particle densities in sucrose versus Ficoll-sucrose gradients
34
DOYLE
AND
COTMAN
TABLE
2
Alkaline Phosphatase Distribution in Sucrose and Ficoll-Sucrose Gradients Containing Synaptosomes Alone, Myelm Alone, and the Actual and Expected Combination of Both Particles Mixed 1: 1 The deviation gradient fraction (A./(A. + B,)) divided into two
of the actual from the expected for a gradient fraction, for example A, is computed by the difference between the actual per cent of total and the expected per cent of total (LB/(& + B,)). Gradients were fractions, above 1.12 gm/cc (A) and below 1.12 gm/cc (B). Pi formed, mNmoles/min/gradient fraction or sample zone
Gradient fraction Sucrose : A B Total Recovery (%I Ficoll-sucrose: A B Total ltecovery (ye) Sample zone
Synaptosomes alone (i )
Myelin alone (3)
Actual combination
Expected combination
Deviation ( yO total)a
-2
0.17 7.14
0.15 0.09
0.42 6.48
0.32 7.23
(48)
6.90 (77)
7.55
(851
0.84 6.84
0.49
0.35
1.00
6.71
0. 13
6.46
(84) 8.58
(96) 0.50
7.46 (84) 8.92
0 These results are representative (within 10yo) alkaline phosphatase distributions were analyzed.
of another
+2
i-2 -2
7.68 9.08
experiment
-2
in which
rather than particle-particle interactions. Firmly established principles explain these differences in particle densities. The buoyant density depends in part on the permeability of the particles to the gradient solute (9,lO). Ficoll-sucrose gradients are essentially isosmotic, and thus do not cause osmotic dehydration which would result in an increase in particle density. Sucrose gradients in the range of concentrations used for isopycnic banding of subcellular particles are hypertonic, and thus promote the dehydration of the particles which makes the particles more dense. As a result, particles with an intravesicular compartment permeable to sucrose, but not to Ficoll, are less dense in Ficoll-sucrose gradients than in sucrose gradients. Day and coworkers (1) also interpret their data based on rate zonal sedimentation in discontinuous sucrose gradients as evidence for particleparticle interactions. We find these data difficult to interpret since measurement of sedimentation changes by light-scattering profiles will
MYELIN-SYNAPTOSOMAL
35
INTERACTIONS
TABLE 3 Cytochrome Oxidase Distribution in Sucrose and Ficoll-Sucrose Gradients Cont’aining Myelin Alone, Synaptosomes Alone, and the Actual and Expected Combination of Both (1: 1) Gradients gdcc
were divided into two fractions:
above 1.12 gm/cc (A) and below
1.12
(B).
Per cent of total cyt,ochrome oxidane Gradient fraction Sucrose : A B Ficoll-sucrose : A B
Myelin alone (4)
Synaptosomes alone ($1
Sctual combinat,ion
0.0 0.0
100.0
100.
0.0 0.0
0. 0 100 0
0.0 100.0
0. 0
Expected combination
0.0 0
0.0 100.0
0. 0 100.0
The data represent results from three experiments. lOOv/, of cytochrome oxidase represents 138 to 318 X 10m4pmole cytochrome c oxidized/min/gradient fraction, where the limit of detectability is 0.5 X 10m4pmole cytochrome c oxidized/min.
not reveal the discrete sedimentation of particular particle populations. We have ourselves analyzed the rate sedimentation of mitochondria and synaptosomes from brain homogenates in a variety of sucrose gradients. Our results could be quantitatively described by the Svedberg equation, which assumes that no particle-particle interactions occur (11). Thus, we find no evidence for myelin-synaptosomal interactions based on analysis of isopycnic or rate zonal centrifugation and conclude that, if such interactions do exist, they must be minimal. SCKNOWLEDGMENT This research was supported and Stroke (NS 08597).
by the National
Institute
of Neurological
Diseases
REFERENCES 1.
E. D.. MCMILLAN, P. N., MICKEY, D. D.. AND APPEL, S. H., Ad. Rioch,em. 39, 29 (1971). 2. MARTIN, R. G., AND AMES, B. N., J. Biol. Chem. 236, 1372 (1961). 3. AUTILIO. I,. A., NORTON, W. T., AND TERRY, R. D., J. Neurochem. 11, 17 (1964). 4. COTMAN, C. W., AND MATTHEWS, D. A., Biochim. Biophys. Acta 249, 380 (1971). 5. EIRBER. E. J., Nut. Cancer Isst. Mono~rq~h 21, 219 (1966). 6. LOWRY, 0. H., ROSEBROUGH. N. J., F~RR, A. I,., .WD RANI)ALL. R. J., J. Biol. Chem. 193, 265 (1951). 7. LOWRY, 0. H., ROBERTS, N. R., WIR. M., HIXON, W. S.. AND CRAMTFORD, E. J., J. Riol. CILem. 207, 19 (1954). DAY.
36
DOYLE
AND
COTMAN
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