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Resolution of components from rat liver homogenate in reorienting density gradients
ANALYTICAL
BIOCHEMISTRY
Resolution
47, 57&583
of Components in
(1972)
from
Rat Liver
Reorienting
Density
0. n-1. GRIFFITH
AND
Receiwd
October
Homogenate
Gradients H. WRIGHT
13. 1971
The technique of reorienting gradients (reograd) for zonal centrifugation was first proposed by Anderson et nl. (1). Prior to this method, the separation of part,icles, ranging in size from whole cells to small protein molecules, was accomplished in the zonal rotor by loading and unloading dynamically through a rotating seal assembly (2). Studies with varying gradient shapes in these dynamically loaded rotors were made. The results showed that the discontinuous gradient was often preferable for the resolution of components from plant and animal tissue homogenates (3,4). In this study the discontinuous gradient was used with the reograd technique to produce resolution similar to that obtained with discontinuous gradients in the dynamically loaded zonal rotors. MATERIALS
$?;D
METHODS
The JCFZ rotor with a specially designed reograd core was used in the model J 21 refrigerated centrifuge. The reograd core is designed with four vanes similar to those of the usual zonal rotor cores. A special screw type cap seal is used for loading and unloading the rotor. This seal is fabricated so that, liquid entering the rotor from the center of the core is directed to the bottom of the rotor through the centrifugal ports of each rotor vane as shown in Fig. 1. As the rotor is filled with gradient (light end first) t.he liquid rises toward the rotor lid and exits through the four holes at the top of the central body of the core. The sample and (if desired) overlay are then pumped in through these same holes using the side port of the cap seal. Both sample and overlay are therefore layered gently on the light or lowest concentration of the gradient. Unloading the rotor is accomplished through displacement by pumping water or air through the side port of the cap seal. The heavy portion of the gradient exits through the center of the rotor core and is monitored by the UV photometer and refractometer. 575 @ 1972
by
Academic
Press,
Inc.
576
FIG.
GRIFFITH
1. Flow
path
of sample
and
grad&t
AND
into
WRIGHT
rotor
through
special
screw
cap seal.
The centrifuge was modified to produce slow acceleration and deceleration between 0 and 3,000 rpm. The maximum volume of the rotor with the core is 1750 ml. Speeds up to 20,000 rpm (40,OOOg) can be attained. The components of the *JCFZ rotor are ehown in Fig. 2A and the assembled rotor is shown in Fig. 2B. -
P‘x.
2A.
Components
of JCFZ
rolor
with
reograd
core
-
and
screw
I
cap seal.
RESOLUTION
FIG.
28.
01”
RAT
Assembled
LIVER
rotor
HOMOGENATE
with
sewn’
577
COMPONIW’L‘S
cap
PP:II a~~:cc:l~ed
Other accessories used in this stdy arc (,(I) the Btel~mn~~ I;V photometer with a 10” recorder and (b) a model 31, Rnuach CCLomb Abbe refractometer. Absorbaucc monitoring of the graclicnt.:: after the run was carried out at a wavelength of 280 mp with :t 1 mm flowthrough curet in the IW 1)hotometer. 3 f-
1“~:. 3. Hypodermic sample and over1a.v
--
--
syringe loading.
-----
attached
io
side
porl
fitling
of screw
cap
seal
fol
578
(GRIFFITH
AND
WRIGHT
All sucrose solut’ions were made from a 66% w/w stock. The method of Cline and Rye1 was adapted for designing the discontinuous gradient and preparing the rat liver homogenate sample (4). The six volumes of sucrose concentrations loaded into the rotor were as follows: 300 ml 8.9% w/w, 300 ml 24.5% w/w, 300 ml 32% w/w, 300 ml 39% w/w, 300 ml 47.0% w/w, and finally 51% w/w unt,il the rotor was filled. The sample volume was 50 ml and the overlay 20 ml ; 4 gm of liver was used in preparing the sample, which was adjusted with buffer to 6% w/w with respect to sucrose concentration. ,411 concentrations were measured by the Abbe refractometer model 3L. The gradient, was loaded into the rot’or by gravity flow at approximately 30 ml/min, after which the sample and overlay were introduced at the top of the gradient by a hypodermic syringe at 10 ml/min as shown in Fig. 3. Before accelerating the loaded rotor, only the special side fitting on the cap seal was removed. The remaining portion of the cap seal was left attached to the rotor. The rotor took 30 min t’o accelerate to 3,000 rpm when the modified slow start for the instrument was employed. After this speed was reached, the instrument was converted to the normal run mode, thus accelerating the rotor to 20,000 r-pm in 6 min. The time at speed for this experiment was 60 min.
1400
FIG. 4A. Profile of six layers of discontinuous unloading of rotor without gradient reorientation. unloading the gradient 45 ml/min.
gradient Flow
with static rate for
loading loading
and and
RESOLUTION
OF RAT
LIVER
HOMOGENATE
COMPONENTS
579
During deceleration the rotor took 5 min to brake from 20,000 to 3,000 rpm, and with the slow deceleration modification 40 min from 3,000 to rest (~‘t = 1.800 X lOlo represents acceleration and deceleration). The rotor was unloaded by pumping water directly through the reattached side port fitting of t’he cap seal. The rotor contents were monitored by the UV photometer and 50 ml fractions were collected for measuring the sucrose concentrat.ions. A plot of these measurements was superimposed on the profile made by t,he UV photometer. RESULTS
Prior to the experiments with the liver homogenate, t’ests were conducted to ascertain the stability of the gradients during loading and unloading the rotor and during reorientation of the gradient. Figure 4A illustrates the shape of the gradient after it was unloaded without reorientation. Figure 4B shows the gradient mlloaded after gradient reorient.ation. In both tests water was used to unload the rotor. Figure 5 shows the resolution of the components from the rat liver homogenate sample. The resolved peaks are similar to those reported by Cline and Rye1 (4). Figure 6 show’ s a similar separat,ion of the same
FIG. 4B. Profile of same gradient shown in Fig. 4A after reorientation. The rotor was loaded at rest with the discontinuous gradient then accelerated to 20,000 rpm for 30 min. Dweleration to rest followed. Static unloading of the gradient at 45 ml/min.
GRIFFITH
Ah-D
WRIGHT
.-..
--j
2.0 1.6
1.0 0.9 i 0.8
I 0.7
-I 0.6
Ed&---a
FIG. 5. Resolution shown
in Figs.
4A and
of components 4B with JCFZ
from rotor
rat and
liver homogenate reograd core.
using
gradient
sample. The discontinuous gradient was made with the Beckman model 141 gradient pump fit,ted with a special discontinuous gradient cam (3). The gradient limits ranged from 9% to 52% w/w. The Ti-15 rotor was used with the B-29 edge unloading liner in the model L3-50 ultracentrifuge. This rotor has a total volume of 1450 ml when the liner is used. The sample (50 ml), overlay (50 ml), and gradient (1,200 ml) were all loaded dynamically at 2,000 rpm. Unloading the separated components
RESOLl-TION
OF RAT
LIVER
HOMOGENATE
581
COMPONENTS
lW-
0.6
0.5
0.4
1
1.3
/l’
4 ‘Id-
1.2
e A b
4
FIG. 6. Resolution of components from aliquot of the same rat liver homogenate sample using Ti-15 rotor with dynamic loading and unloading.
was also at this speed. The run time and speed for this experiment was 30 min at 30,000 rpm (~“t = 1.800 X lOI”). Comparison between the two separations shown in Figs. 5 and 6 was based mainly on particle separation. Identification of the subcellular fractions obtained by zonal centrifugation were reported using electron microscopy, RNA radioactivity, and enzyme activity (5-9). The resolved fractions can therefore be identified (seeFigs. 5 and 6) on
,582
GRIFFITH
AND
WRIGHT
this basis as follows: (a) soluble proteins, (b) free ribosomes, (c) smooth membranes, (d) plasma membranes, (e) mitochondria, and (f) rough endoplasmic reticulum. The shoulder on the right of the soluble proteins may contain soluble RNA as described by El-Aaser et nl. (9). Since our gradient limit was 51% w/w, the separation of cell nuclei and other cell fragments reported elsewhere (10) when using limits above 557% sucrose was not observed. SUMMARY
The subcellular components of rat liver homogenate were separated into seven distinct components when discontinuous gradients were used. The studies reported here demonstrate the similarity of the separation when compared with the work of previous investigators (4). From rest to slow acceleration or deceleration to 3,000 rpm, there is very little sedimentation of the macromolecules in the sample. The additional time required to accelerate t)he reograd rotor when compared to the dynamically loaded rotor does not pose a problem. The hub of the core in the reograd rotor is much larger than in the dynamically loaded rotor; hence the sample is placed in a higher centrifugal field during the initial acceleration. This reduces the dilution or smearing of the sample during the slow acceleration process. Additional sedimentation of the macromolecules due to the lengthy accelerat,ion and deceleration times can be calculated effectively based on the rot,or speed versus the time. A specially designed reorienting gradient core for the zonal rotor was used to permit static loading and unloading. This technique is quite stable for isolating components from plant and animal tissue homogenates. Furthermore, the reograd technique can also be applied to the isopycnic or rate zone separations of selected ribosomes, starch, and especially DNA strands since the sample material is not subjected to the shearing forces of a rotating seal assembly (1). REFERENCES 1. ANDERSON, N. G., PRICE, C. A.. FISHER, W. D., CANNING, R. E.. AND BURGER, C. L., Anal. Biochem. ‘7, 1 (1964). 2. ANDERSON, N. G., J. Phvs. Chem. 66, 1984 (1962). 3. GRIFFITH, 0. M., JACOBSON, Ii. E., CH~FIN, B. R,., DAGG, M. K., AND CLINE, G. B., (1971) unpublished results. 4. CLINE, G. B., AND RYEL, R. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 22, p. 168. Academic Press, New York, 1971. 5. CHAUVEAU, J., MOTILE, Y., ROWLLER. C., AND SCHNEEBELI, J., J. Cell Biol. 12, 17 (1962).
RESOLUTION
OF RAT LIVER
HOMOGENATE
COMPONENTS
583
6. EMMELOT, P., Bus, C. J., BENEDETTI, E. L.. AND Rf~m, PH.. Biochim. Biophys. Acta 90, 126 (1964). 7. LEE, T., SWARTZENDRUBER, D. C.. .~ND SNYDER, F., Biochem. Biophys. Res. Conmun. 36, 748 (1969). 8. SCHLXL. H., SCHUEL, R., AND UNAKAR. N. J., Anal. Biochem. 25, 146 (1968). 9. EL-AASER, A. A.. REID, E., KLUCIS, E., ALEXANDER, P., LETT, J. T., AND SMITH, a.. Xat. Cnncer ILS~. Monogr. 21, 323 (1966). 0. SHEELER, P., GROSS. D. M., .~ND WELLS, J. R., Biochim. Biophvs. Acta 237, 28 (1971).
BIOCHEMISTRY
Resolution
47, 57&583
of Components in
(1972)
from
Rat Liver
Reorienting
Density
0. n-1. GRIFFITH
AND
Receiwd
October
Homogenate
Gradients H. WRIGHT
13. 1971
The technique of reorienting gradients (reograd) for zonal centrifugation was first proposed by Anderson et nl. (1). Prior to this method, the separation of part,icles, ranging in size from whole cells to small protein molecules, was accomplished in the zonal rotor by loading and unloading dynamically through a rotating seal assembly (2). Studies with varying gradient shapes in these dynamically loaded rotors were made. The results showed that the discontinuous gradient was often preferable for the resolution of components from plant and animal tissue homogenates (3,4). In this study the discontinuous gradient was used with the reograd technique to produce resolution similar to that obtained with discontinuous gradients in the dynamically loaded zonal rotors. MATERIALS
$?;D
METHODS
The JCFZ rotor with a specially designed reograd core was used in the model J 21 refrigerated centrifuge. The reograd core is designed with four vanes similar to those of the usual zonal rotor cores. A special screw type cap seal is used for loading and unloading the rotor. This seal is fabricated so that, liquid entering the rotor from the center of the core is directed to the bottom of the rotor through the centrifugal ports of each rotor vane as shown in Fig. 1. As the rotor is filled with gradient (light end first) t.he liquid rises toward the rotor lid and exits through the four holes at the top of the central body of the core. The sample and (if desired) overlay are then pumped in through these same holes using the side port of the cap seal. Both sample and overlay are therefore layered gently on the light or lowest concentration of the gradient. Unloading the rotor is accomplished through displacement by pumping water or air through the side port of the cap seal. The heavy portion of the gradient exits through the center of the rotor core and is monitored by the UV photometer and refractometer. 575 @ 1972
by
Academic
Press,
Inc.
576
FIG.
GRIFFITH
1. Flow
path
of sample
and
grad&t
AND
into
WRIGHT
rotor
through
special
screw
cap seal.
The centrifuge was modified to produce slow acceleration and deceleration between 0 and 3,000 rpm. The maximum volume of the rotor with the core is 1750 ml. Speeds up to 20,000 rpm (40,OOOg) can be attained. The components of the *JCFZ rotor are ehown in Fig. 2A and the assembled rotor is shown in Fig. 2B. -
P‘x.
2A.
Components
of JCFZ
rolor
with
reograd
core
-
and
screw
I
cap seal.
RESOLUTION
FIG.
28.
01”
RAT
Assembled
LIVER
rotor
HOMOGENATE
with
sewn’
577
COMPONIW’L‘S
cap
PP:II a~~:cc:l~ed
Other accessories used in this stdy arc (,(I) the Btel~mn~~ I;V photometer with a 10” recorder and (b) a model 31, Rnuach CCLomb Abbe refractometer. Absorbaucc monitoring of the graclicnt.:: after the run was carried out at a wavelength of 280 mp with :t 1 mm flowthrough curet in the IW 1)hotometer. 3 f-
1“~:. 3. Hypodermic sample and over1a.v
--
--
syringe loading.
-----
attached
io
side
porl
fitling
of screw
cap
seal
fol
578
(GRIFFITH
AND
WRIGHT
All sucrose solut’ions were made from a 66% w/w stock. The method of Cline and Rye1 was adapted for designing the discontinuous gradient and preparing the rat liver homogenate sample (4). The six volumes of sucrose concentrations loaded into the rotor were as follows: 300 ml 8.9% w/w, 300 ml 24.5% w/w, 300 ml 32% w/w, 300 ml 39% w/w, 300 ml 47.0% w/w, and finally 51% w/w unt,il the rotor was filled. The sample volume was 50 ml and the overlay 20 ml ; 4 gm of liver was used in preparing the sample, which was adjusted with buffer to 6% w/w with respect to sucrose concentration. ,411 concentrations were measured by the Abbe refractometer model 3L. The gradient, was loaded into the rot’or by gravity flow at approximately 30 ml/min, after which the sample and overlay were introduced at the top of the gradient by a hypodermic syringe at 10 ml/min as shown in Fig. 3. Before accelerating the loaded rotor, only the special side fitting on the cap seal was removed. The remaining portion of the cap seal was left attached to the rotor. The rotor took 30 min t’o accelerate to 3,000 rpm when the modified slow start for the instrument was employed. After this speed was reached, the instrument was converted to the normal run mode, thus accelerating the rotor to 20,000 r-pm in 6 min. The time at speed for this experiment was 60 min.
1400
FIG. 4A. Profile of six layers of discontinuous unloading of rotor without gradient reorientation. unloading the gradient 45 ml/min.
gradient Flow
with static rate for
loading loading
and and
RESOLUTION
OF RAT
LIVER
HOMOGENATE
COMPONENTS
579
During deceleration the rotor took 5 min to brake from 20,000 to 3,000 rpm, and with the slow deceleration modification 40 min from 3,000 to rest (~‘t = 1.800 X lOlo represents acceleration and deceleration). The rotor was unloaded by pumping water directly through the reattached side port fitting of t’he cap seal. The rotor contents were monitored by the UV photometer and 50 ml fractions were collected for measuring the sucrose concentrat.ions. A plot of these measurements was superimposed on the profile made by t,he UV photometer. RESULTS
Prior to the experiments with the liver homogenate, t’ests were conducted to ascertain the stability of the gradients during loading and unloading the rotor and during reorientation of the gradient. Figure 4A illustrates the shape of the gradient after it was unloaded without reorientation. Figure 4B shows the gradient mlloaded after gradient reorient.ation. In both tests water was used to unload the rotor. Figure 5 shows the resolution of the components from the rat liver homogenate sample. The resolved peaks are similar to those reported by Cline and Rye1 (4). Figure 6 show’ s a similar separat,ion of the same
FIG. 4B. Profile of same gradient shown in Fig. 4A after reorientation. The rotor was loaded at rest with the discontinuous gradient then accelerated to 20,000 rpm for 30 min. Dweleration to rest followed. Static unloading of the gradient at 45 ml/min.
GRIFFITH
Ah-D
WRIGHT
.-..
--j
2.0 1.6
1.0 0.9 i 0.8
I 0.7
-I 0.6
Ed&---a
FIG. 5. Resolution shown
in Figs.
4A and
of components 4B with JCFZ
from rotor
rat and
liver homogenate reograd core.
using
gradient
sample. The discontinuous gradient was made with the Beckman model 141 gradient pump fit,ted with a special discontinuous gradient cam (3). The gradient limits ranged from 9% to 52% w/w. The Ti-15 rotor was used with the B-29 edge unloading liner in the model L3-50 ultracentrifuge. This rotor has a total volume of 1450 ml when the liner is used. The sample (50 ml), overlay (50 ml), and gradient (1,200 ml) were all loaded dynamically at 2,000 rpm. Unloading the separated components
RESOLl-TION
OF RAT
LIVER
HOMOGENATE
581
COMPONENTS
lW-
0.6
0.5
0.4
1
1.3
/l’
4 ‘Id-
1.2
e A b
4
FIG. 6. Resolution of components from aliquot of the same rat liver homogenate sample using Ti-15 rotor with dynamic loading and unloading.
was also at this speed. The run time and speed for this experiment was 30 min at 30,000 rpm (~“t = 1.800 X lOI”). Comparison between the two separations shown in Figs. 5 and 6 was based mainly on particle separation. Identification of the subcellular fractions obtained by zonal centrifugation were reported using electron microscopy, RNA radioactivity, and enzyme activity (5-9). The resolved fractions can therefore be identified (seeFigs. 5 and 6) on
,582
GRIFFITH
AND
WRIGHT
this basis as follows: (a) soluble proteins, (b) free ribosomes, (c) smooth membranes, (d) plasma membranes, (e) mitochondria, and (f) rough endoplasmic reticulum. The shoulder on the right of the soluble proteins may contain soluble RNA as described by El-Aaser et nl. (9). Since our gradient limit was 51% w/w, the separation of cell nuclei and other cell fragments reported elsewhere (10) when using limits above 557% sucrose was not observed. SUMMARY
The subcellular components of rat liver homogenate were separated into seven distinct components when discontinuous gradients were used. The studies reported here demonstrate the similarity of the separation when compared with the work of previous investigators (4). From rest to slow acceleration or deceleration to 3,000 rpm, there is very little sedimentation of the macromolecules in the sample. The additional time required to accelerate t)he reograd rotor when compared to the dynamically loaded rotor does not pose a problem. The hub of the core in the reograd rotor is much larger than in the dynamically loaded rotor; hence the sample is placed in a higher centrifugal field during the initial acceleration. This reduces the dilution or smearing of the sample during the slow acceleration process. Additional sedimentation of the macromolecules due to the lengthy accelerat,ion and deceleration times can be calculated effectively based on the rot,or speed versus the time. A specially designed reorienting gradient core for the zonal rotor was used to permit static loading and unloading. This technique is quite stable for isolating components from plant and animal tissue homogenates. Furthermore, the reograd technique can also be applied to the isopycnic or rate zone separations of selected ribosomes, starch, and especially DNA strands since the sample material is not subjected to the shearing forces of a rotating seal assembly (1). REFERENCES 1. ANDERSON, N. G., PRICE, C. A.. FISHER, W. D., CANNING, R. E.. AND BURGER, C. L., Anal. Biochem. ‘7, 1 (1964). 2. ANDERSON, N. G., J. Phvs. Chem. 66, 1984 (1962). 3. GRIFFITH, 0. M., JACOBSON, Ii. E., CH~FIN, B. R,., DAGG, M. K., AND CLINE, G. B., (1971) unpublished results. 4. CLINE, G. B., AND RYEL, R. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 22, p. 168. Academic Press, New York, 1971. 5. CHAUVEAU, J., MOTILE, Y., ROWLLER. C., AND SCHNEEBELI, J., J. Cell Biol. 12, 17 (1962).
RESOLUTION
OF RAT LIVER
HOMOGENATE
COMPONENTS
583
6. EMMELOT, P., Bus, C. J., BENEDETTI, E. L.. AND Rf~m, PH.. Biochim. Biophys. Acta 90, 126 (1964). 7. LEE, T., SWARTZENDRUBER, D. C.. .~ND SNYDER, F., Biochem. Biophys. Res. Conmun. 36, 748 (1969). 8. SCHLXL. H., SCHUEL, R., AND UNAKAR. N. J., Anal. Biochem. 25, 146 (1968). 9. EL-AASER, A. A.. REID, E., KLUCIS, E., ALEXANDER, P., LETT, J. T., AND SMITH, a.. Xat. Cnncer ILS~. Monogr. 21, 323 (1966). 0. SHEELER, P., GROSS. D. M., .~ND WELLS, J. R., Biochim. Biophvs. Acta 237, 28 (1971).