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Isolation of rat liver microsomes by gel filtration
ANALYTICAL
BIOCHEMISTRY
Isolation
54, 597-603
of Rat Liver 0. TAXGEK,
(1973)
Microsomes
.J. JONSSON,
AND
by Gel
Filtration
S. ORRENIUS
Department of Experimental Medicine, Phnrmacia AB, Uppsala, Sweden, Psychiatric Research Center? Ulleriiker h-o&al, Uppsala, Sweden, and Department of Forertic Medicine, Karolinskn Znstitzctet, Stockholm, Sweden Received
February
28, 1973;
accepted
March
21, 1973
A method was developed for the rapid isolation of liver microsomcs by means of gel filtration. Such microsomes were compared to microsomes prepared by conventional centrifugation techniques. Both preparat,ions vTere of similar composition as regards concentrations and activities of certain microsomal enzymes as well as contamination by other liver cell organelles. The main advantages over conventional techniques are the considerable decrease of time required for isolation of the microsomes, the improved rcmoval of solutes like hemoglobin, the improved suspension stabilitg of the preparation, and that the technique does not. require facilities for ultracentrifugation. IKTRODUCTIOS
The use of microsome suspensions for studies of drug metabolism is steadily increasing (1). However, the conventional centrifugation technique employed for the isolation of microsomes is tedious and time consuming, and an expensive ultracentrifuge is required. Recently, gel filtration has been shown to be a rapid and gentle method for separation of blood platelets from plasma (2). The principle for this separation can readily be applied to the problem of separating microsomes from contaminating solutes in the postmitochondrial supernatant of tissue homogenates as well as in other suspensions. Accordingly, we have investigated the possibility of using gel filtration to isolate liver microsomes. MATERIALS
AND
METHODS
Male Sprague-Dawley rats of body weight 180-250 g were used. The rats were starved for 16 hr prior to decapitation. Livers were removed, blotted, and weighed in ice-cold 0.25 M sucrose. Before homogenization, the livers were cut into small pieces and washed twice in 0.25 M sucrose. The livers from two rats were then homogenized in 2.5 or 5 vol of ice-cold 0.25 M sucrose, and the microsomes prepared as described below in order to prepare 40% and 20% homogenates, respectively. Copyright All rights
597 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.
598
TANGEiY,
JONSSON,
AND
ORRENIUS
Preparation of Control Microsomes. For removal of unbroken cells, nuclei, and cell debris, the homogenate was first centrifuged at 600g for 10 min. The supernatant was carefully decanted and centrifuged for 10 min at 13,300g to remove mitochondria. Ten milliliters of the 13,300g supernatant were gel filtered as described below. The remaining aliquot was centrifuged for 1 hr at 105,006g. After decantation of the supernatant, the microsomal pellet was washed once by resuspension in 0.15 M KC1 and recentrifuged at 105,OOOg for 30 min. The final pellet was homogenized in a 0.15 M KC1/0.05 M potassium phosphate buffer, pH 7.5, and diluted to a final concentration of 0.5 g liver/ml. Preparation of Gel-Filtered Microsomes (GFM) . Sepharose 2B (Pharmacia, Uppsala, Sweden) was washed on a Biichner funnel with 3 vol of acetone followed by large amounts of 0.9% NaCl. Thereafter, the gel was washed and finally equilibrated with the eluant composed of 0.15 M KC1/0.05 M potassium phosphate buffer, pH 7.5, or in some experiments in 0.15 M KC1/0.05 M Tris-HCl buffer, pH 7.5. The gel was suspended in the eluant and poured into the column (Sephadex Laboratory Column K 25/45; 2.5 cm id. X 45 cm). These columns were fitted with gel supporting nylon nets having a pore diameter of 40 /lrn and packed to a height of 20 cm. Ten milliliters of the 13,3OOgsupernatant were carefully layered on the gel under the elution fluid, and the run was started. The eluate was collected by means of a LKB 7000 fraction collector, and 60 drops were collected in each tube. Starting flow rates were in the order of 60 ml/hr. When experiments were performed with a 40% homogenate, half the amount was diluted to 20% and treated as above for control microsomes, the undiluted part being subjected to gel filtrat.ion. Methods of Assay. Protein was determined according to Lowry et al. (3). Aminopyrine demethylase activity was assayed by incubating liver microsomes in a medium consisting of 50 mM potassium phosphate buffer, pH 7.5, 5mM MgCl,, 0.005 mM MnC12, 1 mM NADP+, 5 mM m-isocitrate, 0.4 IU pig heart isocitrate dehydrogenase, and 5 mM aminopyrine for 10 min at 37°C. Formaldehyde was determined as desc.ribed by Nash (4). Isocitrate dehydrogenase activity was measured by omitting aminopyrine and isocitrate dehydrogenase from the incubation medium described above and recording the change of absorbance at 340 nm due to reduction of NADP’. Other methods used have been previously described: for concentration of cytochrome P-450 and hemoglobin (5)) cytochrome b, (6j, and for activities of NADPH-cytochrome c reductase (6)) acid phosphatase (7)) glucose-6-phosphatase (8), catalase (9) and 2- (p-iodophenylj -3- (p-nitrophenylj -5-phenyltetrazolium chloride (INT) -reductase (1Oj.
GEL
FILTRATION
OF
599
MICROSOMES
RESULTS
Gel filtration of the crude 13,300g supernatant resulted in the elution of the microsomal fraction (GFM) in the void volume (V,) within 45 60 min after application to the column. A difference in suspension stability between t.he control and the gel filtered preparations was consistently found in that the cont.roI microsomes tended to sediment upon standing, whereas the GFM remained as a stable suspension. Figure 1 is a representative elution diagram from gel filtration on Sepharose 2B of the 13,300g supcrnatant. The microsomes were elutcd in the first peak absorbing at 280 run. This was demonstrated by the presence of the microsomal marker enzymes, glucose-6-phosphatase and of cytochrome P-450. The second peak contained the solutes of the supernatant. For comparison, cytochrome P-450 difference spect’ra and hemoglobin spectra of the control microsomes and the GFM are shown in Fig. 2. No qualitative difference between the cytochrome P-450 spectra of the two preparations can be seen. In the CO-treated control microsomes, a small, but distinct absorbance at 420 nm demonstrates the presence of measurable hemoglobin in this preparation. No hemoglobin could be detected in the GFM (Fig. 2b). Table 1 summarizes t,he experiments performed on control microsomes and GFM from gel filtration of the 4070 homogenate in order to determine the cont,amination of the two preparations. It can be seen that there were no significant differences in the cytochrome P-450 content per milligram
O0
50 Effluent
volume
loo ImU
150
FIG. 1. Elution diagram of gel filtration on Sepharose 2B of the 13,3009 supernatant from Liver homogenate. The elution pattern of cytochrome P-450 and glucose-6 phosphatase activity demonstrate that the microsomes are eluted in the first peak.
600
TANGEN,
JONSSON,
AND
ORRENIUS
-0.02 ;
-0.01 -0.03 t/, -0.03-
I 420
/, , L / I , 440 460 Wave length (nm)
, I 480
440 460 Wave length (nn,)
480
, I
FIG. 2. Comparison of certain absorption characteristics of control microsomes (a) and of GFM (b). Hemoglobin spectra (dashed lines) were obtained by gassing the sample cuvette with CO for 1 min, and the spectra of cytochrome P-450 (solid lines) after addition of sodium dithionite to both cuvettes. The protein contents werr 0.6 mg/ml and 0.5 mg/ml for the control microsomes and the GFM, respectively.
of protein or in aminopyrine demethylase activity. Contamination by mitochondria, soluble enzymes, lysosomes, and peroxisomes was the same in both preparations as judged by the presence of INT-reductase, isocitrate dehydrogenase, acid phosphatase, and catalase activities. Small amounts of hemoglobin were found in the control microsomes. In contrast, no hemoglobin could be detected in the GFM with the spectrophotometric method used. The yield of microsomal proteins in the GFM was clearly less than in the cont.rols. However, this could be shown to be due to loss of microsomal material during centrifugation of the 40% homogenate and not to the process of gel filtration. This is seen in Table 2, where no significant. differences in the yield of microsomal protein or in the recovery of NADPH-cytochrome c reductase are seen between
GEL
Characteristics
FILTRATION
of Microsomal Gel Filtration
OF
TABLE 1 Fractions Obtained of a 407, Rat Liver
by Ultracentrifugation Homogenate
Microsomal Assay Yield of microsomal protein (mg/g liver) (2)a Cytochrome P-45@ (6) Cytochrome b$ (2) Aminopyrine demethylased Hemoglobinc (6) INT-reductasee (6) Acid phosphatase’ (4) Catalasee (4)
Control
601
MICROSOMES
preparations
microsomesb
GFMb
6.0
13.7
(4)
0.76 + 0.10 0.58 0.0098 + 0.0022 0.07 * 0.04 0.42 + 0.1 0.064 + 0.001 0.54 f 0.19
a Figures in brackets refer to number of experiments. b The figures represent arit,hmetic means +SD, where c nmoles per mg protein. d A absorbance at 412 nm per min per mg protein. e pmoles INT-formazan per hour per mg protein. f pmoles Pi formed per 5 min per mg prot.ein. g A absorbance at 240 nm per min per mg protein.
and
0.72 5 0.19 0.40 0.0110
* 0.0032 0
0.40 k 0.09 0.063 + 0.001 0.68 + 0.06 applicable.
control preparations and GFM from 20% homogenate. Additional evidence demonstrating that the microsome preparations are of equal quality is supplied by the measurements of cytochrome P-450 contents and glucase-6-phosphataseactivities presented in Table 2. DISCUSSION
The isolation and purification of microsomes by means of gel filtration is based on the ability of the Sepharose 2B to separate particulate material from dissolved constituents. This particular type of gel filtration has been discussed by Tangen et al. (2). The limitations of the method are that the particles should be small enough to pass between the gel beads and that the solutes should have molecular weights of less than 24 X 107, which is the range of the exclusion limit for Sepharose2B. The size of the microsomes range from 0.06 to 0.2 km in diameter (11). Consequently, they pass readily through the spaces between the gel beads (t,hese spaces ideally range from 9 to 38 pm in diameter (2) 1. The hydrophilic nat.ure of t.he agarose gel and its almost total lack of electrical charges serve to minimize tailing and damage of the microsomes during gel filtration. The small degree of dilution of the microsomes during gel filtrat,ion and the good preservation of microsomal enzyme act,ivities bear out the validity of these considerations.
602
TANGEN,
Characteristics
of Microsomal Gel Filtration
JONSSON,
AND
ORRENIUS
TABLE 2 Fractions Obtained of a 20y0 Rat Liver
by Ultracentrifugation Homogenate Microsomal
Assay Yield of microsomal protein (mg/gm liver) (3)~ Cytochrome P-450< (6) Glucose-6-phosphatased (3) Acid phosphatase” (3) INT-reductase’ (3) Recovery of NADPH-cyt,ochrome c reductase; percent of homogenate activity (3)
preparat,ion
Cont,rol microsome 18.0 1.07 3.6 0.06 0.45 26.8
f * 5 + i +
and
GFM* 5.0 0.20 0.5 0.02 0.17 1.1
13.4 0.87 4.0 0.06 0.54 25.1
f + + k i f
0.s 0.096 0.1 0.01 0.02 4.7
a Figures in brackets refer to number of experiments. * The figures represent. arithmetic means +SI>. c nmoles per mg protein. d pmoles Pi per 20 min per mg protein. e rmoles Pi per 5 min per mg protein. f pmoles INT-formazan per hr per mg protein.
The contamination of GFM with other subcellular organclles was similar to what was found in the control preparations, as judged by the specific activities of the marker enzymes for mitochondria (INT-reductase) , (catalase) . As these lysosomes (acid phosphatase) , and peroxisomes organelles definitively are larger than t.he exclusion limit of the gel, no separation of these organelles from the microsomes was to be expected. The purity of the GFM in terms of cytochrome P-450 content per milligram of protein and specific activities of aminopyrine demethylase and NADPH-cytochrome c reductase were not significantly different from those of the control microsomes. This implies that separation of the microsomes from contaminating proteins was probably the same in both cases. However, no hemoglobin was found in the GFM in contrast to the control preparation, in which small, but significant amounts of hemoglobin were found. This finding is consistent with a more complete removal of traces of the cytoplasmic solutes by the process of gel filtration. Although it, is often possible to remove all hemoglobin from the liver microsomal fraction by means of further washings and centrifugations or by perfusion of the liver prior to homogenization, these procedures have their specific drawbacks in the additional time required and the possible damage to the microsomes by further centrifugation procedures. Loss of cytochrome P-450 and swelling of the liver during perfusion may also occur (12). Furthermore, there arc other tissues, such as lung, spleen, and
GEL
FILTRATION
OF
MICROSOM
ES
603
fetal liver, where hemoglobin contamination often severely interferes with cytochrome P-450 assays in conventionally isolated microsomes. It is possible that this problem may be minimized or overcome by applying the present technique for the isolation of microsomes from such tissues. Thus, the present work has revealed that it is possible to isolat’e liver microsomes suitable for metabolic studies by gel filtration. The technique is time-saving (more than 1 hr in preparation time is usually saved) and minimizes the cont.aminat’ion of the microsomal fraction with soluble proteins. Like the recently described Ca2+ precipitation technique (13)) it does not require an ultracentrifuge. When further compared to the latter technique, gel filtration reveals the advantage that one avoids aggregation of the microsomes as well as other possible effects on the microsomal membrane by high concentrations of divalent cations. ACKSOWLEDGMENTS This work was supported by the tricentennial fund of the Bank of Sweden. Grant 150 as well as by grants from the Swedish Cancer Society (proj. No. 36-B72-075) and the Swedish Medical Research Council (proj. No. B73-035-2471-06). We thank Mrs. HjSrdis Thor for excellent technical assistance. REFERENCES 1. MANNERING, position & Wilkins
2.
TANGEN.
G. J. (1971) i)~ Fundamentals of Drug Metabolism and Drug Dis(La Du, B. N., Mandel, H. G.. and Way, E. L.. rds.), p. 206. Williams Co.. Baltimore, Maryland. 0.. BERMAN. H. J.. AND MARFEY. P. (1971) Thtwmb. Dint/z. Hnemowh.
25, 268. 3. LOWRY, 0. H., ROSEBROUCH. K;. J., FAHR, A. I,.. ISD RANDALL, R. J. (1951) J. Biol. Chem. 193, 265. 4. XASH, T. (1953) B&hem. 1.55,416. 5. OMURA, T., AND SATO, R. (1964) J. Bid. Chem. 239, 2370. 6. DALLNER, G. (1963) Actn Pnthol. Microbid. Scnr~!. Suppl. 166, 1. 7. BERGMEYER, H. U. (1970) Mrthoden der Enzymatischen Analyse. Band I, 2nd edit.. p. 457, Verlag Chcmie, Weinheim. 8. FISKE, C. H., AND SUBBAROW. J. (1925) J. Biol. Chew 66, 375. 9. BERGMEYER. H. U. (1970) Mcthoden der Enzymatisc~hen Analyse, Band I, 2nd edit., p. 439. Verlag Chemie. Weinheim. 10. PENNINGTON, R. J. (1961) Biochem. J. 80, 649. 11. CLAUDE, A. (1969) itl Microsomcs and Drug Oxidations (Gillette, J. R., Conney, A. M., Cosmides. G. J.. Estabrook. R. W.. Fouts, J. R., rind Mannering, G. J., eds.), p. 3. Academic Press, New York. 12. MAZE, P (1971) in Fundamentals of Drug Metabolism and Drug Disposition (La DLI, B. N., Mandel. H. G.. and Way. E. L.. eds.). p. 527. Williams & Wilkins Co.. Baltimore. Maryland. 13. Ei4MATH. 8. A., AND A~-.~N,~II N.4R.4y.4~, KIi. (1972) A&. Bioc/wv~ 46, 53.
BIOCHEMISTRY
Isolation
54, 597-603
of Rat Liver 0. TAXGEK,
(1973)
Microsomes
.J. JONSSON,
AND
by Gel
Filtration
S. ORRENIUS
Department of Experimental Medicine, Phnrmacia AB, Uppsala, Sweden, Psychiatric Research Center? Ulleriiker h-o&al, Uppsala, Sweden, and Department of Forertic Medicine, Karolinskn Znstitzctet, Stockholm, Sweden Received
February
28, 1973;
accepted
March
21, 1973
A method was developed for the rapid isolation of liver microsomcs by means of gel filtration. Such microsomes were compared to microsomes prepared by conventional centrifugation techniques. Both preparat,ions vTere of similar composition as regards concentrations and activities of certain microsomal enzymes as well as contamination by other liver cell organelles. The main advantages over conventional techniques are the considerable decrease of time required for isolation of the microsomes, the improved rcmoval of solutes like hemoglobin, the improved suspension stabilitg of the preparation, and that the technique does not. require facilities for ultracentrifugation. IKTRODUCTIOS
The use of microsome suspensions for studies of drug metabolism is steadily increasing (1). However, the conventional centrifugation technique employed for the isolation of microsomes is tedious and time consuming, and an expensive ultracentrifuge is required. Recently, gel filtration has been shown to be a rapid and gentle method for separation of blood platelets from plasma (2). The principle for this separation can readily be applied to the problem of separating microsomes from contaminating solutes in the postmitochondrial supernatant of tissue homogenates as well as in other suspensions. Accordingly, we have investigated the possibility of using gel filtration to isolate liver microsomes. MATERIALS
AND
METHODS
Male Sprague-Dawley rats of body weight 180-250 g were used. The rats were starved for 16 hr prior to decapitation. Livers were removed, blotted, and weighed in ice-cold 0.25 M sucrose. Before homogenization, the livers were cut into small pieces and washed twice in 0.25 M sucrose. The livers from two rats were then homogenized in 2.5 or 5 vol of ice-cold 0.25 M sucrose, and the microsomes prepared as described below in order to prepare 40% and 20% homogenates, respectively. Copyright All rights
597 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.
598
TANGEiY,
JONSSON,
AND
ORRENIUS
Preparation of Control Microsomes. For removal of unbroken cells, nuclei, and cell debris, the homogenate was first centrifuged at 600g for 10 min. The supernatant was carefully decanted and centrifuged for 10 min at 13,300g to remove mitochondria. Ten milliliters of the 13,300g supernatant were gel filtered as described below. The remaining aliquot was centrifuged for 1 hr at 105,006g. After decantation of the supernatant, the microsomal pellet was washed once by resuspension in 0.15 M KC1 and recentrifuged at 105,OOOg for 30 min. The final pellet was homogenized in a 0.15 M KC1/0.05 M potassium phosphate buffer, pH 7.5, and diluted to a final concentration of 0.5 g liver/ml. Preparation of Gel-Filtered Microsomes (GFM) . Sepharose 2B (Pharmacia, Uppsala, Sweden) was washed on a Biichner funnel with 3 vol of acetone followed by large amounts of 0.9% NaCl. Thereafter, the gel was washed and finally equilibrated with the eluant composed of 0.15 M KC1/0.05 M potassium phosphate buffer, pH 7.5, or in some experiments in 0.15 M KC1/0.05 M Tris-HCl buffer, pH 7.5. The gel was suspended in the eluant and poured into the column (Sephadex Laboratory Column K 25/45; 2.5 cm id. X 45 cm). These columns were fitted with gel supporting nylon nets having a pore diameter of 40 /lrn and packed to a height of 20 cm. Ten milliliters of the 13,3OOgsupernatant were carefully layered on the gel under the elution fluid, and the run was started. The eluate was collected by means of a LKB 7000 fraction collector, and 60 drops were collected in each tube. Starting flow rates were in the order of 60 ml/hr. When experiments were performed with a 40% homogenate, half the amount was diluted to 20% and treated as above for control microsomes, the undiluted part being subjected to gel filtrat.ion. Methods of Assay. Protein was determined according to Lowry et al. (3). Aminopyrine demethylase activity was assayed by incubating liver microsomes in a medium consisting of 50 mM potassium phosphate buffer, pH 7.5, 5mM MgCl,, 0.005 mM MnC12, 1 mM NADP+, 5 mM m-isocitrate, 0.4 IU pig heart isocitrate dehydrogenase, and 5 mM aminopyrine for 10 min at 37°C. Formaldehyde was determined as desc.ribed by Nash (4). Isocitrate dehydrogenase activity was measured by omitting aminopyrine and isocitrate dehydrogenase from the incubation medium described above and recording the change of absorbance at 340 nm due to reduction of NADP’. Other methods used have been previously described: for concentration of cytochrome P-450 and hemoglobin (5)) cytochrome b, (6j, and for activities of NADPH-cytochrome c reductase (6)) acid phosphatase (7)) glucose-6-phosphatase (8), catalase (9) and 2- (p-iodophenylj -3- (p-nitrophenylj -5-phenyltetrazolium chloride (INT) -reductase (1Oj.
GEL
FILTRATION
OF
599
MICROSOMES
RESULTS
Gel filtration of the crude 13,300g supernatant resulted in the elution of the microsomal fraction (GFM) in the void volume (V,) within 45 60 min after application to the column. A difference in suspension stability between t.he control and the gel filtered preparations was consistently found in that the cont.roI microsomes tended to sediment upon standing, whereas the GFM remained as a stable suspension. Figure 1 is a representative elution diagram from gel filtration on Sepharose 2B of the 13,300g supcrnatant. The microsomes were elutcd in the first peak absorbing at 280 run. This was demonstrated by the presence of the microsomal marker enzymes, glucose-6-phosphatase and of cytochrome P-450. The second peak contained the solutes of the supernatant. For comparison, cytochrome P-450 difference spect’ra and hemoglobin spectra of the control microsomes and the GFM are shown in Fig. 2. No qualitative difference between the cytochrome P-450 spectra of the two preparations can be seen. In the CO-treated control microsomes, a small, but distinct absorbance at 420 nm demonstrates the presence of measurable hemoglobin in this preparation. No hemoglobin could be detected in the GFM (Fig. 2b). Table 1 summarizes t,he experiments performed on control microsomes and GFM from gel filtration of the 4070 homogenate in order to determine the cont,amination of the two preparations. It can be seen that there were no significant differences in the cytochrome P-450 content per milligram
O0
50 Effluent
volume
loo ImU
150
FIG. 1. Elution diagram of gel filtration on Sepharose 2B of the 13,3009 supernatant from Liver homogenate. The elution pattern of cytochrome P-450 and glucose-6 phosphatase activity demonstrate that the microsomes are eluted in the first peak.
600
TANGEN,
JONSSON,
AND
ORRENIUS
-0.02 ;
-0.01 -0.03 t/, -0.03-
I 420
/, , L / I , 440 460 Wave length (nm)
, I 480
440 460 Wave length (nn,)
480
, I
FIG. 2. Comparison of certain absorption characteristics of control microsomes (a) and of GFM (b). Hemoglobin spectra (dashed lines) were obtained by gassing the sample cuvette with CO for 1 min, and the spectra of cytochrome P-450 (solid lines) after addition of sodium dithionite to both cuvettes. The protein contents werr 0.6 mg/ml and 0.5 mg/ml for the control microsomes and the GFM, respectively.
of protein or in aminopyrine demethylase activity. Contamination by mitochondria, soluble enzymes, lysosomes, and peroxisomes was the same in both preparations as judged by the presence of INT-reductase, isocitrate dehydrogenase, acid phosphatase, and catalase activities. Small amounts of hemoglobin were found in the control microsomes. In contrast, no hemoglobin could be detected in the GFM with the spectrophotometric method used. The yield of microsomal proteins in the GFM was clearly less than in the cont.rols. However, this could be shown to be due to loss of microsomal material during centrifugation of the 40% homogenate and not to the process of gel filtration. This is seen in Table 2, where no significant. differences in the yield of microsomal protein or in the recovery of NADPH-cytochrome c reductase are seen between
GEL
Characteristics
FILTRATION
of Microsomal Gel Filtration
OF
TABLE 1 Fractions Obtained of a 407, Rat Liver
by Ultracentrifugation Homogenate
Microsomal Assay Yield of microsomal protein (mg/g liver) (2)a Cytochrome P-45@ (6) Cytochrome b$ (2) Aminopyrine demethylased Hemoglobinc (6) INT-reductasee (6) Acid phosphatase’ (4) Catalasee (4)
Control
601
MICROSOMES
preparations
microsomesb
GFMb
6.0
13.7
(4)
0.76 + 0.10 0.58 0.0098 + 0.0022 0.07 * 0.04 0.42 + 0.1 0.064 + 0.001 0.54 f 0.19
a Figures in brackets refer to number of experiments. b The figures represent arit,hmetic means +SD, where c nmoles per mg protein. d A absorbance at 412 nm per min per mg protein. e pmoles INT-formazan per hour per mg protein. f pmoles Pi formed per 5 min per mg prot.ein. g A absorbance at 240 nm per min per mg protein.
and
0.72 5 0.19 0.40 0.0110
* 0.0032 0
0.40 k 0.09 0.063 + 0.001 0.68 + 0.06 applicable.
control preparations and GFM from 20% homogenate. Additional evidence demonstrating that the microsome preparations are of equal quality is supplied by the measurements of cytochrome P-450 contents and glucase-6-phosphataseactivities presented in Table 2. DISCUSSION
The isolation and purification of microsomes by means of gel filtration is based on the ability of the Sepharose 2B to separate particulate material from dissolved constituents. This particular type of gel filtration has been discussed by Tangen et al. (2). The limitations of the method are that the particles should be small enough to pass between the gel beads and that the solutes should have molecular weights of less than 24 X 107, which is the range of the exclusion limit for Sepharose2B. The size of the microsomes range from 0.06 to 0.2 km in diameter (11). Consequently, they pass readily through the spaces between the gel beads (t,hese spaces ideally range from 9 to 38 pm in diameter (2) 1. The hydrophilic nat.ure of t.he agarose gel and its almost total lack of electrical charges serve to minimize tailing and damage of the microsomes during gel filtration. The small degree of dilution of the microsomes during gel filtrat,ion and the good preservation of microsomal enzyme act,ivities bear out the validity of these considerations.
602
TANGEN,
Characteristics
of Microsomal Gel Filtration
JONSSON,
AND
ORRENIUS
TABLE 2 Fractions Obtained of a 20y0 Rat Liver
by Ultracentrifugation Homogenate Microsomal
Assay Yield of microsomal protein (mg/gm liver) (3)~ Cytochrome P-450< (6) Glucose-6-phosphatased (3) Acid phosphatase” (3) INT-reductase’ (3) Recovery of NADPH-cyt,ochrome c reductase; percent of homogenate activity (3)
preparat,ion
Cont,rol microsome 18.0 1.07 3.6 0.06 0.45 26.8
f * 5 + i +
and
GFM* 5.0 0.20 0.5 0.02 0.17 1.1
13.4 0.87 4.0 0.06 0.54 25.1
f + + k i f
0.s 0.096 0.1 0.01 0.02 4.7
a Figures in brackets refer to number of experiments. * The figures represent. arithmetic means +SI>. c nmoles per mg protein. d pmoles Pi per 20 min per mg protein. e rmoles Pi per 5 min per mg protein. f pmoles INT-formazan per hr per mg protein.
The contamination of GFM with other subcellular organclles was similar to what was found in the control preparations, as judged by the specific activities of the marker enzymes for mitochondria (INT-reductase) , (catalase) . As these lysosomes (acid phosphatase) , and peroxisomes organelles definitively are larger than t.he exclusion limit of the gel, no separation of these organelles from the microsomes was to be expected. The purity of the GFM in terms of cytochrome P-450 content per milligram of protein and specific activities of aminopyrine demethylase and NADPH-cytochrome c reductase were not significantly different from those of the control microsomes. This implies that separation of the microsomes from contaminating proteins was probably the same in both cases. However, no hemoglobin was found in the GFM in contrast to the control preparation, in which small, but significant amounts of hemoglobin were found. This finding is consistent with a more complete removal of traces of the cytoplasmic solutes by the process of gel filtration. Although it, is often possible to remove all hemoglobin from the liver microsomal fraction by means of further washings and centrifugations or by perfusion of the liver prior to homogenization, these procedures have their specific drawbacks in the additional time required and the possible damage to the microsomes by further centrifugation procedures. Loss of cytochrome P-450 and swelling of the liver during perfusion may also occur (12). Furthermore, there arc other tissues, such as lung, spleen, and
GEL
FILTRATION
OF
MICROSOM
ES
603
fetal liver, where hemoglobin contamination often severely interferes with cytochrome P-450 assays in conventionally isolated microsomes. It is possible that this problem may be minimized or overcome by applying the present technique for the isolation of microsomes from such tissues. Thus, the present work has revealed that it is possible to isolat’e liver microsomes suitable for metabolic studies by gel filtration. The technique is time-saving (more than 1 hr in preparation time is usually saved) and minimizes the cont.aminat’ion of the microsomal fraction with soluble proteins. Like the recently described Ca2+ precipitation technique (13)) it does not require an ultracentrifuge. When further compared to the latter technique, gel filtration reveals the advantage that one avoids aggregation of the microsomes as well as other possible effects on the microsomal membrane by high concentrations of divalent cations. ACKSOWLEDGMENTS This work was supported by the tricentennial fund of the Bank of Sweden. Grant 150 as well as by grants from the Swedish Cancer Society (proj. No. 36-B72-075) and the Swedish Medical Research Council (proj. No. B73-035-2471-06). We thank Mrs. HjSrdis Thor for excellent technical assistance. REFERENCES 1. MANNERING, position & Wilkins
2.
TANGEN.
G. J. (1971) i)~ Fundamentals of Drug Metabolism and Drug Dis(La Du, B. N., Mandel, H. G.. and Way, E. L.. rds.), p. 206. Williams Co.. Baltimore, Maryland. 0.. BERMAN. H. J.. AND MARFEY. P. (1971) Thtwmb. Dint/z. Hnemowh.
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